Thermoplastic Nanofluidic Devices for Identifying Abasic Sites in Single DNA Molecules

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Steven A Soper ◽  
Swarnagowri Vaidyanathan ◽  
Franklin Uba ◽  
Bo Hu ◽  
David Kaufman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Therapeutic Efficacy ◽  
Double Strand Breaks ◽  
Dna Molecules ◽  
Strand Breaks ◽  
Single Dna Molecules ◽  
Abasic Sites ◽  
Nanofluidic Devices ◽  
Ap Sites

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy...

scholarly journals Accumulation of Endogenous DNA Damage Sustain Malignant Progression and Increase Therapy Resistance in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1788-1788
Author(s):  
Maria Gkotzamanidou ◽  
Evangelos Terpos ◽  
Nikhil C. Munshi ◽  
Vassilis L. Souliotis ◽  
Meletios A. Dimopoulos
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Research Funding ◽  
Progressive Increase ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Oncology Research ◽  
Ap Sites

Introduction: Increased endogenous DNA damage pose a serious threat to cell health, since it may lead to mutagenesis, genomic instability and cellular apoptosis. Therefore, a better understanding of the molecular mechanisms implicated in the formation and repair of the endogenous DNA damage can be exploited for understanding pathogenesis and progression of cancer, as well as for new treatment opportunities. Herein, we report high levels of endogenous DNA damage in multiple myeloma (MM) and characterize its type. Methods: Malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of 16 patients with monoclonal gammopathy of undetermined significance (MGUS; 9F/7M; median age, 65 years; range, 43-87 years), 18 with smoldering myeloma (SMM; 10F/8M; median age, 63 years; range, 37-88 years) and 41 with MM (19F/22M; median age, 65 years; range, 34-79 years) were analyzed. Patients with MM were categorized according to their outcome to responders (≥PR, n=27) and non-responders (n=14) to subsequent melphalan therapy. PBMCs from 40 healthy controls (HC) were examined in parallel. In untreated cells, the endogenous DNA damage (using alkaline comet assay), the detection of critical markers of the DNA damage response and repair (DDR/R) network (using western blot), basal oxidative stress (using a luminescence-based assay) and the endogenous levels of abasic sites (AP-sites) were analyzed. Moreover, cells were ex vivo treated with 100μg/ml melphalan for 5min and critical DDR/R signals, including fundamental DNA repair mechanisms [nucleotide excision repair (NER) using Southern blot; interstrand cross-links repair (ICL/R) using quantitative-PCR; double-strand breaks repair (DSB/R) using immunofluorescence quantification of γH2AX foci by confocal microscopy] and the induction of apoptosis (using a photometric enzyme-immunoassay) were evaluated. The study was approved by the Institutional review board of Alexandra Hospital and all subjects provided informed consent. The study was conducted according to the Declaration of Helsinki. Results: In both BMPCs and PBMCs from MGUS, SMM and MM patients, significant progressive increase in the endogenous single-strand breaks (SSBs) and double-strand breaks (DSBs) was found in MGUS, SMM and MM patients, respectively; PBMCs from HC showed the lowest DNA damage levels (all P<0.05). Interestingly, MM patients, non-responders to subsequent melphalan therapy showed significantly elevated levels of endogenous DNA damage compared with responders (P<0.001). Also, in both tissues analyzed, significant progressive increase in the induction of molecular markers associated with DSBs (γH2AX, p-53BP1) and SSBs (p-RPA32) was observed in MGUS, SMM and MM patients, respectively, confirming the progressive accumulation of both SSBs and DSBs in patients' cells. Moreover, in both BMPCs and PBMCs, significant progressive increase in the oxidative stress and the AP-sites was observed in MGUS, SMM and MM patients; PBMCs from HC showed the lowest levels of both factors analyzed (all P<0.05). Importantly, non-responders MM patients exhibited significantly higher levels of oxidative stress and AP-sites compared with responders (P<0.05). Significant correlations were observed between the endogenous DNA damage and the oxidative stress or the AP-sites in the same patients, suggesting that the endogenous DNA damage may, at least partly, result from oxidative stress and/or the induction of AP-sites. In addition, in both tissues analyzed, substantial progressive increase in the NER, ICL/R and DSB/R efficiencies were observed in MGUS, SMM and MM patients, respectively; PBMCs from HC showed the lowest repair rates (all P<0.001). Interestingly, non-responders MM patients showed higher NER, ICL/R and DSB/R rates than responders (P<0.001). Finally, in both BMPCs and PBMCs a gradual suppression of the apoptotic pathway was observed in MGUS, SMM and MM patients; PBMCs from HC showed the highest apoptosis rates (all P<0.001). Interestingly, non-responders MM patients showed significantly lower apoptosis rates compared to responders (P<0.001). Conclusion: Increased levels of endogenously generated DNA damage, which are correlated with increased formation of DNA damage and preserved DNA repair, may be implicated in the transformation process of myelomagenesis and the outcome of anti-myeloma therapy. Disclosures Terpos: Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Medison: Honoraria. Munshi:Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Adaptive: Consultancy. Dimopoulos:Sanofi Oncology: Research Funding.

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 PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Deepti Sharma ◽  
Louis De Falco ◽  
Sivaraman Padavattan ◽  
Chang Rao ◽  
Susana Geifman-Shochat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleosome Core ◽  
Epigenetic Marker ◽  
Kinetics Of

AbstractThe poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

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 Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

scholarly journals Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

2019 ◽  
Vol 116 (39) ◽  
pp. 19552-19562 ◽  
Author(s):  
Justine Sitz ◽  
Sophie Anne Blanchet ◽  
Steven F. Gameiro ◽  
Elise Biquand ◽  
Tia M. Morgan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Genomic Instability ◽  
E3 Ligase ◽  
Human Papillomaviruses ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

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