scholarly journals Persistent DNA Double-Strand Breaks After Repeated Diagnostic CT Scans in Breast Epithelial Cells and Lymphocytes

2021 ◽  
Vol 11 ◽  
Author(s):  
Natalia V. Bogdanova ◽  
Nina Jguburia ◽  
Dhanya Ramachandran ◽  
Nora Nischik ◽  
Katharina Stemwedel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Cell Types ◽  
Strand Break ◽  
Ct Scans ◽  
Breast Epithelial Cell ◽  
Damage Repair ◽  
Breast Epithelial ◽  
The Impact ◽  
Diagnostic Ct

DNA double-strand break (DSB) induction and repair have been widely studied in radiation therapy (RT); however little is known about the impact of very low exposures from repeated computed tomography (CT) scans for the efficiency of repair. In our current study, DSB repair and kinetics were investigated in side-by-side comparison of RT treatment (2 Gy) with repeated diagnostic CT scans (≤20 mGy) in human breast epithelial cell lines and lymphoblastoid cells harboring different mutations in known DNA damage repair proteins. Immunocytochemical analysis of well known DSB markers γH2AX and 53BP1, within 48 h after each treatment, revealed highly correlated numbers of foci and similar appearance/disappearance profiles. The levels of γH2AX and 53BP1 foci after CT scans were up to 30% of those occurring 0.5 h after 2 Gy irradiation. The DNA damage repair after diagnostic CT scans was monitored and quantitatively assessed by both γH2AX and 53BP1 foci in different cell types. Subsequent diagnostic CT scans in 6 and/or 12 weeks intervals resulted in elevated background levels of repair foci, more pronounced in cells that were prone to genomic instability due to mutations in known regulators of DNA damage response (DDR). The levels of persistent foci remained enhanced for up to 6 months. This “memory effect” may reflect a radiation-induced long-term response of cells after low-dose x-ray exposure.

scholarly journals Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260852
Author(s):  
Meryem Ozgencil ◽  
Julian Barwell ◽  
Marc Tischkowitz ◽  
Louise Izatt ◽  
Ian Kesterton ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Ips Cells ◽  
Variants Of Uncertain Significance ◽  
Cellular Models ◽  
Pathogenic Variants ◽  
Damage Repair ◽  
Uncertain Significance ◽  
Induced Pluripotent ◽  
The Impact

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Effect of enzalutamide on sensitivity in prostate cancer cells to radiation by inhibition of DNA double strand break repair.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 208-208 ◽  
Author(s):  
Maryam Ghashghaei ◽  
Thierry Muanza ◽  
Miltiadis Paliouras ◽  
Tamim Niazi
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Dna Damage Repair ◽  
Strand Break ◽  
Dependent Manner ◽  
Castration Resistance ◽  
Concurrent Administration ◽  
Damage Repair ◽  
Pathway Inhibition ◽  
Approved Drugs

208 Background: Prostate cancer is the second leading cause of cancer-related deaths amongst men in North America. Data suggests that, following radiation therapy (XRT), androgen receptor (AR) enhances DNA damage repair and contributes to resistance of prostate cancer (PCa) cells to XRT. At present AR-pathway inhibition is the mainstay treatment of metastatic castration resistance prostate cancer (mCRPC). Enzalutamide (ENZA), a potent AR inhibitor is one of the approved drugs in this setting. The purpose of this study was to assess the potential radiosensitization of ENZA and its mechanism of action in hormone resistant PCa cells. Methods: The effect of ENZA alone or in combination with XRT was assessed on hormone-sensitive, (HS: LNCaP, PC3-T877A) and insensitive PCa cells (HI: PC3, PC3-AR V7, C4-2) using viability and clonogenic assays, cell cycle arrest and DNA damage analysis. Results: MTT assay demonstrates that ENZA significantly inhibits the proliferation of HS PCa cells in a dose dependent manner whereas CRPC required ENZA in combination with ADT (androgen deprivation therapy). Additionally, clonogenic assay proves that concurrent administration of ENZA or ADT+ENZA and XRT led to a supra-additive antitumor effect with the dose enhancement factor of 1.76±0.008 in LNCaP, 1.65±0.01 in PC3-T877A and 1.35±0.003 in C4-2 respectively at surviving fraction of 0.1. This effect was not observed in PC3 and PC3-AR V7 cells pre-treated with ENZA (in all cases DEF = 1 at SF = 0.1). Additionally, the level of γH2AX increased in HS cells and CRPC cells treated with ENZA/ADT+ENZA and XRT when compared to XRT alone. The enhanced H2AX activity remained unchanged up to 24 hours after combination treatment. Furthermore, there is an initial inhibition of DNA-PKcs in HS and CRPC cells treated with ENZA/ADT+ENZA administered before XRT. Conclusions: Our data suggest that the higher efficacy of ENZA/ENZA+ADT and XRT could be partially due to inhibition of DNA damage repair. Our results demonstrated a significant enhancement of XRT efficacy and confirms the rational for the ongoing combination clinical trials with XRT.

scholarly journals Recruitment of the Type B Histone Acetyltransferase Hat1p to Chromatin Is Linked to DNA Double-Strand Breaks

2006 ◽  
Vol 26 (9) ◽  
pp. 3649-3658 ◽  
Author(s):  
Song Qin ◽  
Mark R. Parthun
Keyword(s):  
Dna Damage ◽  
Molecular Mechanisms ◽  
Dna Damage Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Similar Distribution ◽  
Tail Domain ◽  
Type B ◽  
Direct Role ◽  
Damage Repair

ABSTRACT Type B histone acetyltransferases are thought to catalyze the acetylation of the NH2-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH2-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.

scholarly journals PARP inhibition in Ewing sarcoma: impact of germline DNA damage repair defects and activation of immunoregulatory pathways

2020 ◽  
Author(s):  
Lisa M. Maurer ◽  
Rosemarie E. Venier ◽  
Elina Mukherjee ◽  
Claire M. Julian ◽  
Jessica D. Daley ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ewing Sarcoma ◽  
Immune Checkpoint ◽  
Dna Damage Repair ◽  
Germline Mutations ◽  
Parp Inhibition ◽  
Damage Repair ◽  
The Impact ◽  
Ewing Tumor

ABSTRACTEwing sarcoma, an oncofusion-driven primary bone tumor, can occur in the setting of various germline mutations in DNA damage repair pathway genes. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. PARP inhibitors (PARPi) are effective against Ewing sarcoma cells in vitro, though have demonstrated limited success in clinical trials to date. In order to assess the impact of BARD1 loss on Ewing sarcoma sensitivity to PARP inhibitor therapy, we generated the novel PSaRC318 patient-derived Ewing tumor cell from our patient with a germline BARD1 mutation and then analyzed the response of these cells to PARPi. We demonstrate that PSaRC318 cells are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that loss of BARD1 enhances PARPi sensitivity. In certain malignancies, DNA damage can activate the IRF1 (interferon response factor 1) immunoregulatory pathway, and the activation of this pathway can drive immunosuppression through upregulation of the immune checkpoint protein PD-L1. In order to determine the ability of PARPi to alter Ewing tumor immunoregulation, we evaluated whether PARPi results in upregulation of the IRF1-PDL1 pathway. Indeed, we now demonstrate that PARPi leads to increased PD-L1 expression in Ewing sarcoma. Together, these data thus far suggest that while Ewing tumors harboring germline mutations in DNA damage repair proteins may in respond to PARPi in vitro, in vivo benefit of PARPi may only be demonstrated when counteracting the immunosuppressive effects of DNA damage by concurrently targeting immune checkpoint proteins.

scholarly journals Dual RNA 3’-end processing of H2A.X messenger RNA maintains DNA damage repair throughout the cell cycle

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Esther Griesbach ◽  
Margarita Schlackow ◽  
William F. Marzluff ◽  
Nick J. Proudfoot
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Messenger Rna ◽  
De Novo ◽  
Dna Damage Repair ◽  
Cell Types ◽  
Histone Variants ◽  
Histone Genes ◽  
Mrna Isoforms ◽  
Damage Repair

AbstractPhosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3’-end processing in the maintenance of effective DDR.

scholarly journals Targeting BRCA and DNA Damage Repair Genes in GI Cancers: Pathophysiology and Clinical Perspectives

2021 ◽  
Vol 11 ◽  
Author(s):  
Kai Zimmer ◽  
Florian Kocher ◽  
Alberto Puccini ◽  
Andreas Seeber
Keyword(s):  
Dna Damage ◽  
Clinical Investigation ◽  
Dna Damage Repair ◽  
Cost Effective ◽  
Therapeutic Implications ◽  
Damage Repair ◽  
New Treatment ◽  
The Impact ◽  
Repair Genes ◽  
Breast Cancer Gene

Mutated germline alleles in the DNA damage repair (DDR) genes “breast cancer gene 1” (BRCA1) and BRCA2 have originally been identified as major susceptibility genes in breast and ovarian cancers. With the establishment and approval of more cost-effective gene sequencing methods, germline and somatic BRCA mutations have been detected in several cancers. Since the approval of poly (ADP)-ribose polymerase inhibitors (PARPi) for BRCA-mutated cancers, BRCA mutations gained rising therapeutic implications. The impact and significance of BRCA mutations have been evaluated extensively in the last decades. Moreover, other genes involved in the DDR pathway, such as ATM, ATR, or CHK1, have emerged as potential new treatment targets, as inhibitors of these proteins are currently under clinical investigation. This review gives a concise overview on the emerging clinical implications of mutations in the DDR genes in gastrointestinal cancers with a focus on BRCA mutations.

scholarly journals DNA Damage Repair Gene Mutations Are Indicative of a Favorable Prognosis in Colorectal Cancer Treated With Immune Checkpoint Inhibitors

2021 ◽  
Vol 10 ◽  
Author(s):  
Yipeng Song ◽  
Jian Huang ◽  
Dandan Liang ◽  
Ying Hu ◽  
Beibei Mao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Damage ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Dna Damage Repair ◽  
Genomic Data ◽  
Cancer Center ◽  
Damage Repair ◽  
The Impact

BackgroundDNA damage repair (DDR) genes were recently implicated in the anti-tumor immune response. Therefore, it is worthwhile to unravel the implications of DDR pathways in the shaping of immune responsiveness in colorectal cancer (CRC) patients receiving immune checkpoint inhibitors (ICI).MethodsWe analyzed publicly available genomic data from a cohort treated with ICI from Memorial Sloan Kettering Cancer Center (MSK ICI cohort). To characterize the impact of the DDR mutation, the genomic data of The Cancer Genome Atlas (TCGA) colorectal adenocarcinoma (COADREAD) dataset was explored. We also analyzed the incidence of DDR mutation and microsatellite instability-high (MSI-H) in a Chinese CRC cohort using panel sequencing.ResultsThe DDR pathway was commonly mutated (21.8%) in the multicancer MSK ICI cohort, with the highest frequency of 36.4% in CRCs. Survival analysis showed that DDR mutation correlated with an improved overall survival (OS) in CRCs and pan-cancer in the MSK ICI cohort. However, no significant associations were identified in the TCGA COADREAD and MSK non-ICI CRCs. DDR mutation was associated with higher tumor mutational burden (TMB) levels and increased immune cell infiltration and immune checkpoint molecule expression in the TCGA COADREAD dataset. Last, we investigated the DDR mutational pattern and its associations with MSI-H and other genomic features in a Chinese CRC cohort. Notably, MSI-H and DDR mutation was present in 5.7% and 13.4% of cases, respectively, which suggests that DDR identifies a higher proportion of potential responders than MSI-H.ConclusionOur data suggest that DDR mutation as an indication of enhanced cancer immunity, and it may function as a biomarker for patients with CRCs receiving ICI treatment. The high incidence of DDR mutation in the Chinese CRC cohort emphasizes the future utility of panel-based DDR evaluation in guiding ICI treatment.

scholarly journals Ablating Ku70 phosphorylation results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma

2021 ◽  
Author(s):  
Janapriya Saha ◽  
Jinsung Bae ◽  
Shih-Ya Wang ◽  
Lori J. Chappell ◽  
Purva Gopal ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Damage ◽  
Genomic Instability ◽  
Dna Damage Repair ◽  
Strand Break ◽  
Damage Repair ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Transformed Cell Lines ◽  
Dna Ends

SUMMARYMultiple pathways mediate the repair of DNA double-strand break (DSB), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that phosphorylation of the non-homologous end joining (NHEJ) factor, Ku70, is required for the dissociation of the Ku heterodimer from DNA ends to allow DSB repair via homologous recombination (HR). A knock-in mouse, in which phosphorylation is ablated in the three conserved sites of Ku70 (Ku703A/3A), was generated in order to test the hypothesis that Ku70 phosphorylation is required for initiation of HR and that blocking this process results in enhanced genomic instability and tumorigenesis. Here, we show that Ku703A/3A mice develop spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting DNA repair is decreased in these mice. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that phosphorylation-mediated dissociation of Ku heterodimer from DNA ends is required for efficient DNA damage repair and disruption of this process results in genomic instability and accelerated development of HCC.

Abstract 125: Proteome Analysis of a Regulatory Pathway of Pathologic Vascular Injury as Mediated by KLF5 and its Transcriptional Complexes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Kenichi Aizawa ◽  
Toru Suzuki ◽  
Takayoshi Mastumura ◽  
Nanae Kada ◽  
Daigo Sawaki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Proteome Analysis ◽  
Strand Break ◽  
Double Strand Break ◽  
Chain Gene ◽  
Repair System ◽  
Peptide Mass ◽  
Damage Repair ◽  
A Chain

Background: Transcription factor Krüppel-like factor 5 (KLF5) is a key element linking external stress and cardiovascular remodeling by up-regulating platelet derived growth factor (PDGF)-A chain gene activity. However, the underlying mechanisms remain to be elucidated. The unambiguous and comprehensive identification of interacting proteins is crucial for understanding these mechanisms. In the present study, we identified interacting factors of KLF5 by proteomic analysis and characterized their regulation in the vascular pathogenic response. Methods&Results: Double-stranded oligonucleotide containing the binding sequence for KLF5 in the PDGF-A promoter was synthesized and attached to metal beads, to which cell nuclear extract was applied. SDS-PAGE visualized specific bands to the sequence, which were subjected to in-gel digestion and peptide mass fingerprinting by MALDI-TOF/MS spectrometry. Factors that are known to be important in the DNA damage/repair pathway were successively identified. We therefore examined the involvement of the complex in vascular pathologies. Double-strand break as determined by immunohistochemistry using γ-H2AX antibody, a marker of activation of the double-stranded DNA damage/repair response, was observed in pathogenically stimulated vascular endothelial cells (HUVEC) and neointimal tissues in rat carotid artery balloon injury model. Further, KLF5 was shown to mediate the response on γ-H2AX as shown by co-immunoprecipitation and confocal microscopy. Discussion: We show a hitherto unknown regulatory mechanism by DNA double-strand break/repair system involving KLF5 in the vascular pathogenic response. Our findings might provide a clue to understanding the initiation of pathological cell proliferation observed in atherosclerosis or restenosis after coronary intervention. This new pathway might also be a tempting target for therapeutic intervention aimed at modulating the activity of KLF5 upon PDGF-A chain and its associated pathologies in the cardiovascular system.

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