scholarly journals Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo

2020 ◽  
Vol 48 (17) ◽  
pp. 9694-9709
Author(s):  
Zhengping Shao ◽  
Brian J Lee ◽  
Élise Rouleau-Turcotte ◽  
Marie-France Langelier ◽  
Xiaohui Lin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Live Cell Imaging ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Downstream Effector ◽  
Mutation Analyses ◽  
Molecular Nature

Abstract DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as ‘trapping’. To understand the molecular nature of ‘trapping’ in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

Histone marks: repairing DNA breaks within the context of chromatin

2012 ◽  
Vol 40 (2) ◽  
pp. 370-376 ◽  
Author(s):  
Kyle M. Miller ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genome Stability ◽  
Nuclear Dna ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Dsbs

Inherited or acquired defects in detecting, signalling or repairing DNA damage are associated with various human pathologies, including immunodeficiencies, neurodegenerative diseases and various forms of cancer. Nuclear DNA is packaged into chromatin and therefore the true in vivo substrate of damaged DNA occurs within the context of chromatin. Our work aims to decipher the mechanisms by which cells detect DNA damage and signal its presence to the DNA-repair and cell-cycle machineries. In particular, much of our work has focused on DNA DSBs (double-strand breaks) that are generated by ionizing radiation and radiomimetic chemicals, and which can also arise when the DNA replication apparatus encounters other DNA lesions. In the present review, we describe some of our recent work, as well as the work of other laboratories, that has identified new chromatin proteins that mediate DSB responses, control SDB processing or modulate chromatin structure at DNA-damage sites. We also aim to survey several recent advances in the field that have contributed to our understanding of how particular histone modifications and involved in DNA repair. It is our hope that by understanding the role of chromatin and its modifications in promoting DNA repair and genome stability, this knowledge will provide opportunities for developing novel classes of drugs to treat human diseases, including cancer.

scholarly journals Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2352-2366 ◽  
Author(s):  
Guo-zhong Yi ◽  
Guanglong Huang ◽  
Manlan Guo ◽  
Xi’an Zhang ◽  
Hai Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Progression Free Survival ◽  
Recurrent Glioblastoma ◽  
Dna Lesions ◽  
Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

Drug-induced DNA damage and tumor chemosensitivity.

1990 ◽  
Vol 8 (12) ◽  
pp. 2062-2084 ◽  
Author(s):  
R J Epstein
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Tumor Cell ◽  
Malignant Disease ◽  
Dna Lesions ◽  
Drug Induced ◽  
Cell Subpopulations ◽  
Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

PARP inhibitors for cancer therapy

2005 ◽  
Vol 7 (4) ◽  
pp. 1-20 ◽  
Author(s):  
Nicola J. Curtin
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Alkylating Agents ◽  
Parp Inhibitors ◽  
Ionising Radiation ◽  
Parp Inhibition ◽  
Tumour Regression ◽  
Dna Breaks ◽  
Parp 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a zinc-finger DNA-binding enzyme that is activated by binding to DNA breaks. Poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 converts DNA damage into intracellular signals that activate either DNA repair by the base-excision pathway or cell death. A family of 18 PARPs has been identified, but only the most abundant, PARP-1 and PARP-2, which are both nuclear enzymes, are activated by DNA damage. PARP inhibitors of ever-increasing potency have been developed in the 40 years since the discovery of PARP-1, both as tools for the investigation of PARP-1 function and as potential modulators of DNA-repair-mediated resistance to cytotoxic therapy. Owing to the high level of homology between the catalytic domains of PARP-1 and PARP-2, the inhibitors probably affect both enzymes. Convincing biochemical evidence, which has been corroborated by genetic manipulation of PARP-1 activity, shows that PARP inhibition is associated with increased sensitivity to DNA-alkylating agents, topoisomerase I poisons and ionising radiation. Novel PARP inhibitors of sufficient potency and suitable pharmacokinetic properties to allow evaluation in animal models have been shown to enhance the antitumour activity of temozolomide (a DNA-methylating agent), topoisomerase poisons and ionising radiation; indeed, the combination with temozolomide resulted in complete tumour regression in two independent studies. The combination of a PARP inhibitor and temozolomide is currently undergoing clinical evaluation for the first time.

scholarly journals Poly(ADP-ribose)-binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions

2020 ◽  
Author(s):  
Ramhari Kumbhar ◽  
Jullian Perren ◽  
Fade Gong ◽  
David Corujo ◽  
Frank Medina ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
Histone Demethylase ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Binding Region ◽  
Transcriptional Responses ◽  
Dna Lesion ◽  
Two Factors

AbstractThe histone demethylase KDM5A removes histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDR). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a non-canonical poly(ADP-ribose), (PAR), binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and functions at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR-binding and macroH2A1.2 in KDM5A recognition of damage sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.SummaryThe histone demethylase KDM5A demethylates H3K4 to promote repair and transcriptional responses at DNA breaks. We identified poly(ADP-ribose)-binding and macroH2A1.2 as modulators of KDM5A association with DNA damage sites, revealing how KDM5A engages DNA breaks within chromatin.

scholarly journals ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sonia Jimeno ◽  
Rosario Prados-Carvajal ◽  
María Jesús Fernández-Ávila ◽  
Sonia Silva ◽  
Domenico Alessandro Silvestris ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Rna Editing ◽  
Genomic Stability ◽  
Strand Break ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Dna Double Strand Break ◽  
Editing Enzyme

AbstractThe maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

scholarly journals Analysis of DNA breaks, DNA damage response, and apoptosis produced by high NaCl

2008 ◽  
Vol 295 (6) ◽  
pp. F1678-F1688 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Maurice B. Burg
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Hela Cells ◽  
Cell Types ◽  
Dna Breaks ◽  
Main Subject ◽  
Mrn Complex ◽  
Damage Response

We previously reported that, both in cell culture and in the renal inner medulla in vivo, elevating NaCl increased the number of DNA breaks, which persisted as long as NaCl remained high but were rapidly repaired when NaCl was lowered. Furthermore, those breaks did not induce the DNA repair protein γH2AX or cause activation of the MRN (Mre11, Rad50, Nbs1) complex. In contrast, others recently reported that high NaCl does induce γH2AX and MRN complex formation and concluded that these activities are associated with repair of the DNA (Sheen MR, Kim SW, Jung JY, Ahn JY, Rhee JG, Kwon HM, Woo SK. Am J Physiol Renal Physiol 291: F1014–F1020, 2006). The purpose of the present studies was to resolve the disparity. The important difference is that HeLa cells, which were the main subject of the later report, are much less tolerant of high NaCl than are the mIMCD3 cells, which were our main subject. mIMCD3 cells survive levels of NaCl that kill HeLa cells by apoptosis. Here we demonstrate that in both cell types raising NaCl to a level that the cells survive (higher for mIMCD3 than HeLa) increases DNA breaks without inducing γH2AX or activating the MRN complex and that the DNA breaks persist as long as NaCl remains elevated, but are rapidly repaired when it is lowered. Importantly, in both cell types, raising NaCl further to cause apoptosis activates these DNA damage response proteins and greatly fragments DNA, associated with cell death. We conclude that γH2AX induction and MRN activation in response to high NaCl are associated with apoptosis, not DNA repair.

scholarly journals Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Mahara Valverde ◽  
Jonathan Lozano-Salgado ◽  
Paola Fortini ◽  
Maria Alexandra Rodriguez-Sastre ◽  
Emilio Rojas ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Stem Cells ◽  
Dna Damage ◽  
Dna Repair ◽  
Mesenchymal Stem Cells ◽  
Adipocyte Differentiation ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Repair Capacity ◽  
Dna Repair Capacity

Human adipose-derived mesenchymal stem cells (hADMSCs) are recognized as a potential tool in cell tissue therapy because of their capacity to proliferate and differentiate in vitro. Several studies have addressed their use in regenerative medicine; however, little is known regarding their response to DNA damage and in particular to the reactive oxygen species (ROS) that are present in the microenvironment of implantation. In this study, we used the ROS-inducing agent hydrogen peroxide to explore the responses of (1) hADMSCs and (2) derived terminally differentiated adipocytes to oxidatively generated DNA damage. Using single cell gel electrophoresis, a dose-related increase was found for both DNA breaks and oxidative lesions (formamidopyrimidine DNA glycosylase-sensitive sites) upon exposure of hADMSCs to hydrogen peroxide. DNA repair capacity of hADMSCs was affected in cells exposed to 150 and 200 μM of hydrogen peroxide. An increase in the basal levels of DNA breaks and oxidative DNA lesions was observed through adipocyte differentiation. In addition, hydrogen peroxide-induced DNA damage increased through adipocyte differentiation; DNA repair capacity also decreased. This study is the first follow-up report on DNA repair capacity during adipogenic differentiation. Remarkably, in terminally differentiated adipocytes, DNA breakage repair is abolished while the repair of DNA oxidative lesions remains efficient.

scholarly journals Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1284
Author(s):  
Anzhela V. Pavlova ◽  
Elena A. Kubareva ◽  
Mayya V. Monakhova ◽  
Maria I. Zvereva ◽  
Nina G. Dolinnaya
Keyword(s):  
Dna Damage ◽  
Genome Instability ◽  
Critical Role ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Dna Breaks ◽  
Dna Damage And Repair ◽  
Repair Processes

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.

Inhibition of Poly ADP Ribose Polymerase (PARP) Activity Exerts Cytotoxic Effects on Chromosomal Instability Syndrome and Leukaemic Cell Lines: Potential for Anti-Leukaemia Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2647-2647
Author(s):  
Terry J. Gaymes ◽  
S. Shall ◽  
Farzin Farzaneh ◽  
Ghulam J. Mufti
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Survival ◽  
Cell Lines ◽  
Chromosomal Instability ◽  
Parp Inhibitors ◽  
Dna Breaks ◽  
Leukaemic Cells ◽  
Parp Activity

Abstract Recent reports suggest that BrCA1−/− and BrCA2−/− cells can be selectively targeted for cell death through abrogation of their PARP activity. It is postulated that as a result of PARP inhibition, accumulation of single strand DNA breaks (SSB) leads to the replication fork collapse and conversion of SSB to double strand DNA breaks (DSB). The inability of repair defective cells such as BrCA2−/− to repair the DSB would lead to cell death. Exploitation of DNA repair defects using PARP inhibitors (PI) thus represents a more specific and less toxic form of therapy for a number of haematological malignancies. Chromosomal instability (CI) syndromes that have inherent defects in double strand DNA repair also have a uniformly high incidence of transformation to acute leukaemia or lymphoma. In order to test the efficacy of PI therapy we analysed CI cell lines, myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) cell lines and the potential for combination therapy with inhibitors of DNA methyltransferase (DNMTi) or histone deacetylase inhibitors (HDACi). We report that cells from CI syndromes; Blooms syndrome, Fanconi Anaemia (FancD2 and FancA), Ataxia telancgectasia and Nijmegen break syndrome display abnormal cell cycle profiles and excessive apoptosis in response to the PI’s PJ34 (3μM) and EB47 (45μM). In contrast, normal control cells displayed standard cell cycle profiles and no apoptosis in response to PI at equivalent concentrations. Clonogenic cytotoxicity assays showed that CI syndrome cells exhibit between 30–75% cell survival compared with 100% cell survival in control cells (p<0.05) in response to PI. The homologous recombination (HR) DNA repair component, rad51 forms foci in response to DNA damage. In HR compromised cells, rad51 foci fail to form. In response to PI, immunofluorescent studies show that CI syndrome cells demonstrate severely reduced rad51 foci formation (<5%) compared to control cells (15%). This confirms that PI targets the HR deficiencies in CI syndrome cells. Histone γH2AX, phosphorylated in response to DSB had greatly increased foci formation in CI syndrome cells compared to control cells as a result of unrepaired DNA damage (25.3 vs 9.3%)(p<0.05). CI syndromes have increased transformation potential to the MDS and AML. Addition of 3μM PJ34 to the myelomonocytoid leukaemic/myelodysplastic cell line, P39 exhibited significant apoptosis, with a cell survival fraction of 65% compared to 100% in control cells (p<0.01). Immunofluorescent studies revealed reduced rad51 foci formation (6.3 vs 15%) and increased γH2AX foci formation (17.6 vs 9.3%)(p<0.01). Strikingly, we were also able to reproduce similar PI responses in the Jurkat T-cell leukaemic cell line. We next explored the use of PI in combination with DNMTi or HDACi. Whilst 3μM PJ34 offered only additive effects on decitabine cytotoxicity, a sub-optimal concentration (1μM) of PJ34 behaved synergistically with HDACi potentiating the cytotoxic effect of 200nM MS275 by 55% compared to MS275 alone (p<0.05) in P39 cells. In conclusion, we have shown that in a panel of CI syndrome and leukaemic cells, PI demonstrates significant cytotoxic responses. We also show that PI acts synergistically in combination with HDACi. Parp inhibitors can potentially exploit DSB repair defects in leukaemic cells paving the way for a targeted therapy for MDS and leukaemia.

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