scholarly journals Duplex-Repair enables highly accurate sequencing, despite DNA damage

2021 ◽  
Author(s):  
Kan Xiong ◽  
Douglas Shea ◽  
Justin Rhoades ◽  
Timothy Blewett ◽  
Ruolin Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Accuracy ◽  
Base Pairs ◽  
Free Dna ◽  
Duplex Sequencing ◽  
Tumor Biopsies ◽  
The Impact ◽  
Formalin Fixed ◽  
Fixed Tumor

Abstract Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.

scholarly journals Duplex-Repair enables highly accurate sequencing, despite DNA damage

2021 ◽  
Author(s):  
Kan Xiong ◽  
Doug Shea ◽  
Justin Rhoades ◽  
Tim Blewett ◽  
Ruolin Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Accuracy ◽  
Base Pairs ◽  
Free Dna ◽  
Duplex Sequencing ◽  
Tumor Biopsies ◽  
The Impact ◽  
Formalin Fixed ◽  
Fixed Tumor

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via End Repair/dA-Tailing, may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior duplex base pairs from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles heel in sequencing and offers a solution to restore high accuracy.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus

2014 ◽  
Vol 191 ◽  
pp. 145-150 ◽  
Author(s):  
Zoran Gačić ◽  
Stoimir Kolarević ◽  
Karolina Sunjog ◽  
Margareta Kračun-Kolarević ◽  
Momir Paunović ◽  
...  
Keyword(s):  
Dna Damage ◽  
Freshwater Mussels ◽  
In Vitro Exposure ◽  
Unio Tumidus ◽  
The Impact ◽  
Unio Pictorum

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.

Induction of the DNA damage response by IAP inhibition triggers natural immunity via upregulation of NKG2D ligands in Hodgkin lymphoma in vitro

2013 ◽  
Vol 394 (10) ◽  
pp. 1325-1331 ◽  
Author(s):  
Maike Sauer ◽  
Katrin S. Reiners ◽  
Hinrich P. Hansen ◽  
Andreas Engert ◽  
Stephan Gasser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hodgkin Lymphoma ◽  
Dna Damage Response ◽  
Natural Immunity ◽  
Immune Recognition ◽  
Iap Antagonist ◽  
Damage Response ◽  
The Impact ◽  
Apoptosis Proteins

Abstract Evasion of apoptosis is a hallmark of cancer cells. Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of programmed cell death and are overexpressed in several tumors including Hodgkin lymphoma (HL). Preclinical studies indicate antitumor activity of IAP antagonists and clinical studies in hematological malignancies are underway. Here, we investigate the impact of the small molecule IAP antagonist LCL161 on HL cell lines. Although the antagonist caused rapid degradation of cIAP1 leading to TNFα secretion, LCL161 did not promote apoptosis significantly. However, LCL161 induced expression of MICA and MICB, ligands for the activating immune receptor NKG2D, and enhanced the susceptibility of HL cells to NKG2D-dependent lysis by NK cells. MICA/B upregulation was dependent on activation of the DNA damage response upon LCL161 treatment. Taken together, we demonstrate a novel link between IAP inhibition, DNA damage and immune recognition.

P–437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Bailie ◽  
M Maidarti ◽  
R Hawthorn ◽  
S Jack ◽  
N Watson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Histological Analysis ◽  
High Dose ◽  
Follicle Growth ◽  
Repair Capacity ◽  
Testosterone Undecanoate ◽  
The Impact ◽  
Transgender Men

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture. What is known already: Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool. Study design, size, duration: Whole ovaries were obtained from four transgender men aged 25–36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12–16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26–36). Participants/materials, setting, methods: Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed. Main results and the role of chance: At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p < 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p < 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p < 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p < 0.005). After culture, γH2AX expression increased in both transgender (p < 0.005) and controls (p < 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p < 0.05). At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p < 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p < 0.05) and a less marked decline in RAD51 expression(p < 0.05). The expression of MRE–11 in control non-growing oocytes dramatically declined (100% to 58.2%, p < 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings: These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures. Trial registration number n/a

Cellular Senescence Contributes to the Outcome of Lymphoma Chemotherapy by Cell-Autonomous and Paracrine Immune Mechanisms

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3617-3617
Author(s):  
Jan Dörr ◽  
Yong Yu ◽  
Bernd Dörken ◽  
Clemens A. Schmitt
Keyword(s):  
Dna Damage ◽  
Immune Cell ◽  
Genetic Alterations ◽  
Cell Interactions ◽  
Brdu Incorporation ◽  
Tumor Site ◽  
Immune Cell Subsets ◽  
The Impact

Abstract Introduction: Premature senescence reflects an acutely inducible, irreversible growth arrest as a cellular response to stresses such as oncogenic activation and DNA damage, including chemotherapeutic anticancer agents. Senescence complements apoptosis as a tumor suppressive and therapeutic effector principle, but whether a selective disruption of the senescence machinery impairs treatment outcome is unknown. Moreover, function and fate of senescent tumor cells within the tumor site remain unclear. Here, we analyze the impact of defined genetic alterations, i.e. Bcl2 overexpression (blocking apoptosis), deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence), and conditional expression of p53 (mediating both apoptosis and senescence), on therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model with specific emphasis on immunological tumor-host and growth-modulating senescent/non-senescent cell interactions as a consequence of TIS in vitro and in vivo. Methods: Lymphoma cells (LCs) of various genetic backgrounds were retrovirally transduced with the bcl2 gene to study TIS in the absence of drug-induced apoptosis. Bcl2-protected LCs were treated with the DNA damaging anticancer agent adriamycin in vitro, or were exposed to the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. TIS was detected by staining for senescence-associated β-galactosidase activity (SA-β-gal) and other senescence-related markers, including Ki67 and BrdU incorporation. To study tumor-host cell interactions, isolated normal splenocytes were co-incubated with proliferating or senescent LCs in vitro. Immunophenotyping was carried out with antibodies specific for macrophages, granulocytes, natural killer cells and T-lymphocytes. Cytokine production was measured by protein arrays. Results: Senescent LCs engage in cell-cell interactions with different immune cell subsets, in particular macrophages, granulocytes and T-cells in vitro. Fluorescence microscopy reveals that macrophages engulf LCs after they entered TIS. In vivo, TIS correlates with the quantitative attraction of immune cell populations to the tumor site and subsequent clearing of senescent cells. Ongoing mechanistic studies on underlying ligand/receptor interactions will be reported at the meeting. TIS cells exhibit a specific pro-inflammatory secretory profile whose functional impact on tumor and bystander cells is currently being investigated. Importantly, this profile is distinguishable from cytokine profiles of senescence-compromised Suv39h1- or p53-deficient lymphomas, and, thus, reflects a senescence - rather than a DNA damage-associated secretory response. Discussion: The study unveils a functional interaction of senescent LCs with different immune cell subsets in vitro and in vivo. The cytokine arrays show that senescent cells produce a specific secretory profile, which might stimulate immune cell attraction. Therefore, immune cells could be recruited to lymphomas in vivo specifically after TIS with the potential to clear senescent – and possibly non-senescent – cells from the tumor site. The data demonstrate genetically that senescence is a beneficial effector principle of DNA damaging chemotherapy and encourage further exploration of this program to limit cancer expansion in vivo.

scholarly journals Acute Toxicity and DNA Instability Induced by Exposure to Low Doses of Triclosan and Phthalate DEHP, and Their Combinations, in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Nathalia de Assis Aguilar Duarte ◽  
Lindiane Eloisa de Lima ◽  
Flora Troina Maraslis ◽  
Michael Kundi ◽  
Emilene Arusievicz Nunes ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Toxicity ◽  
Nuclear Division ◽  
Aquatic Organisms ◽  
Human Cells ◽  
Dna Instability ◽  
Nuclear Division Index ◽  
Mutagenic Effects ◽  
The Impact

Triclosan (TCS) is an antimicrobial agent widely used in personal care products (PCP) and the di-(2-ethyl hydroxy-phthalate) (DEHP) is a chemical compound derived from phthalic acid, used in medical devices and plastic products with polyvinyl chloride (PVCs). As result of their extensive use, TCS and DEHP have been found in the environment and previous studies demonstrated the association between their exposure and toxic effects, mostly in aquatic organisms, but there is a shortage in the literature concerning the exposure of TCS and DEHP in human cells. The aim of the present study was to assess the impact of exposure to TCS and DEHP, as well as their combinations, on biomarkers related to acute toxicity and DNA instability, in HepG2 cells, by use of cytokinesis-block micronucleus cytome (CBMNCyt) assay. For that, the cultures were exposed to TCS, DEHP and combinations at doses of 0.10, 1.0, and 10 μM for the period of 4 h and the parameters related to DNA damage (i.e., frequencies of micronuclei (MN) and nuclear buds (NBUDs), to cell division (i.e., nuclear division index (NDI) and nuclear division cytotoxic index (NDCI) and to cell death (apoptotic and necrotic cells) were scored. Clear mutagenic effects were seen in cells treated with TCS, DEHP at doses of 1.0 and 10 μM, but no combined effects were observed when the cells were exposed to the combinations of TCS + DEHP. On the other hand, the combination of the toxicants significantly increased the frequencies of apoptotic and necrotic cells, as well as induced alterations of biomarkers related to cell viability (NDI and NDCI), when compared to the groups treated only with TCS or DEHP. Taken together, the results showed that TCS and DEHP are also able to induce acute toxicity and DNA damage in human cells.

P-437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Bailie ◽  
M Maidarti ◽  
R Hawthorn ◽  
S Jack ◽  
N Watson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Histological Analysis ◽  
High Dose ◽  
Follicle Growth ◽  
Repair Capacity ◽  
Testosterone Undecanoate ◽  
The Impact ◽  
Transgender Men

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture.  What is known already Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool Study design, size, duration Whole ovaries were obtained from four transgender men aged 25-36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12-16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26-36).  Participants/materials, setting, methods Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed.  Main results and the role of chance At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p < 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p < 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p < 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p < 0.005). After culture, γH2AX expression increased in both transgender (p < 0.005) and controls (p < 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p < 0.05).  At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p < 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p < 0.05) and a less marked decline in RAD51 expression(p < 0.05). The expression of MRE-11 in control non-growing oocytes dramatically declined (100% to 58.2%, p < 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures.  Trial registration number n/a

Sign in / Sign up

Export Citation Format

Share Document