No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

1986 ◽  
Vol 166 (2) ◽  
pp. 497-509 ◽  
Author(s):  
Peter J. Mayer ◽  
Matthews O. Bradley ◽  
Warren W. Nichols
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts

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 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.

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

scholarly journals Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Richard I Tuxworth ◽  
Matthew J Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

Abstract DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals Attenuating the DNA damage response to double strand breaks restores function in models of CNS neurodegeneration

2018 ◽  
Author(s):  
Richard I. Tuxworth ◽  
Matthew J. Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

AbstractDNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.

1996 ◽  
Vol 135 (5) ◽  
pp. 1369-1376 ◽  
Author(s):  
V V Didenko ◽  
P J Hornsby
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Dna Fragments ◽  
Strand Breaks ◽  
Single Base ◽  
Double Stranded Dna

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.

scholarly journals JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 709-718 ◽  
Author(s):  
Tanyel Kiziltepe ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Enrique M. Ocio ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Endonuclease G ◽  
Human Multiple Myeloma

Abstract Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO•) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K–induced cytotoxicity was mediated via NO• in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH2-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K–induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K–induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

scholarly journals Induction and repair of DNA double-strand breaks in hippocampal neurons of miсe of different age after exposure to 60Со γ-rays in vivo and in vitro

2018 ◽  
Vol 177 ◽  
pp. 06001
Author(s):  
R.A. Kozhina ◽  
V.N. Chausov ◽  
E.A. Kuzmina ◽  
A.V. Boreyko
Keyword(s):  
Central Nervous System ◽  
Dna Damage ◽  
Nervous System ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Age Related ◽  
Γ Rays

One of the central problems of modern radiobiology is the study of DNA damage induction and repair mechanisms in central nervous system cells, in particular, in hippocampal cells. The study of the regularities of molecular damage formation and repair in the hippocampus cells is of special interest, because these cells, unlike most cells of the central nervous system (CNS), keep proliferative activity, i.e. ability to neurogenesis. Age-related changes in hippocampus play an important role, which could lead to radiosensitivity changes in neurons to the ionizing radiation exposure. Regularities in DNA double-strand breaks (DSB) induction and repair in different aged mice hippocampal cells in vivo and in vitro under the action of γ-rays 60Со were studied with DNA comet-assay. The obtained dose dependences of DNA DSB induction are linear both in vivo and in vitro. It is established that in young animals' cells, the degree of DNA damage is higher than in older animals. It is shown that repair kinetics is basically different for exposure in vivo and in vitro.

scholarly journals DNA Double Strand Breaks in the Toxoplasma Gondii-Infected Cells by the Action of Reactive Oxygen Species

2020 ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Yi Yang ◽  
Xueqiu Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background: Toxoplasma gondii (T. gondii) is an obligate parasite of the warm-blooded animals with a worldwide distribution. Once having entered a host cell, it manipulates host’s DNA damage response that is yet to be investigated. The objectives of the present study were three-fold: 1) to assess DNA damages in T. gondii-infected cells in vitro; 2) to ascertain sources causing DNA damage in T. gondii-infected cells; 3) to investigate activation of DNA damage response during T. gondii infection.Methods: HeLa, Vero and HEK293 cells were infected with T. gondii at multiplicity of infection (MOI) of 10:1. Infected cells at 10 h, 20 h or 30 h post infection were analyzed for a DNA double strand breaks (DSBs) biomarker γH2AX using Western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were examined using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), and the impact of ROS on DNA damage was assessed by inhibition using a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage response in these T. gondii-infected cells was evaluated by detecting the expression of active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) with Western blot. Results: Compared to uninfected cells, γH2AX expression in the infected HeLa cells at 10 h, 20 h, and 30 h was increased over time during T. gondii infection. NAC treatment reduced ROS level in host cells and significantly decreased the expression of γH2AX. Expression of phosphorylated ATM/CHK2 was elevated in T. gondii-infected cells.Conclusion: T. gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also concomitantly activated DNA damage response pathway ATM/CHK2. T. gondii struggles a balance between survival and apoptosis of its host cells for the benefit of its own survival.

Sign in / Sign up

Export Citation Format

Share Document