scholarly journals DNA Strand Break Properties of Protoporphyrin IX by X-ray Irradiation against Melanoma

2020 ◽  
Vol 21 (7) ◽  
pp. 2302 ◽  
Author(s):  
Takema Hasegawa ◽  
Junko Takahashi ◽  
Shinsuke Nagasawa ◽  
Motomichi Doi ◽  
Akihiro Moriyama ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dissolved Oxygen ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Strand Break ◽  
Ros Generation ◽  
Strand Breaks ◽  
X Ray

Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3′-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O2•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.

scholarly journals Recombinogenic Flap Ligation Pathway for Intrinsic Repair of Topoisomerase IB-Induced Double-Strand Breaks

2000 ◽  
Vol 20 (21) ◽  
pp. 8059-8068 ◽  
Author(s):  
Chonghui Cheng ◽  
Stewart Shuman
Keyword(s):  
High Efficiency ◽  
Strand Break ◽  
Strand Transfer ◽  
Strand Breaks ◽  
Genomic Deletions ◽  
Topoisomerase Ib ◽  
Dna Strand ◽  
Recombination Pathway

ABSTRACT Topoisomerase IB catalyzes recombinogenic DNA strand transfer reactions in vitro and in vivo. Here we characterize a new pathway of topoisomerase-mediated DNA ligation in vitro (flap ligation) in which vaccinia virus topoisomerase bound to a blunt-end DNA joins the covalently held strand to a 5′ resected end of a duplex DNA containing a 3′ tail. The joining reaction occurs with high efficiency when the sequence of the 3′ tail is complementary to that of the scissile strand immediately 5′ of the cleavage site. A 6-nucleotide segment of complementarity suffices for efficient flap ligation. Invasion of the flap into the duplex apparently occurs while topoisomerase remains bound to DNA, thereby implying a conformational flexibility of the topoisomerase clamp around the DNA target site. The 3′ flap acceptor DNA mimics a processed end in the double-strand-break-repair recombination pathway. Our findings suggest that topoisomerase-induced breaks may be rectified by flap ligation, with ensuing genomic deletions or translocations.

scholarly journals DNA double-strand break repair: a theoretical framework and its application

2016 ◽  
Vol 13 (114) ◽  
pp. 20150679 ◽  
Author(s):  
Philip J. Murray ◽  
Bart Cornelissen ◽  
Katherine A. Vallis ◽  
S. Jon Chapman
Keyword(s):  
Dna Damage ◽  
Auger Electron ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

scholarly journals βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

2019 ◽  
Vol 27 (4) ◽  
pp. 1200-1213 ◽  
Author(s):  
Ainhoa Nieto ◽  
Makoto R. Hara ◽  
Victor Quereda ◽  
Wayne Grant ◽  
Vanessa Saunders ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Molecular Scaffold ◽  
Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

In vitro and in vivo effects of HAL on porphyrin production in rat bladder cancer cells (AY27)

2019 ◽  
Vol 23 (07n08) ◽  
pp. 813-820
Author(s):  
Odrun A. Gederaas ◽  
Harald Husebye ◽  
Anders Johnsson ◽  
Susan Callaghan ◽  
Anders Brunsvik
Keyword(s):  
Bladder Cancer ◽  
Cancer Cells ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Active Agent ◽  
Laser Scanning Microscopy ◽  
Rat Bladder ◽  
Bladder Cancer Cells

Aminolevulinic acid and hexyl-aminolevulinate serve as biological precursors to produce photosensitive porphyrins in cells via the heme biosynthetic pathway. This pathway is integral to porphyrin-based photodynamic diagnosis and therapy. By adding exogenous hexyl-aminolevulinate to rat bladder cancer cells (AY27, in vitro) and an animal bladder cancer model (in vivo), fluorescent endogenous porphyrin production was stimulated. Lipophilic protoporphyrin IX was identified as the dominant species by reverse high-pressure liquid chromatography. Subcellular porphyrin localization in the AY27 cells was evaluated by confocal laser scanning microscopy and showed almost quantitative bleaching after 20 s. From this study, we ascertained that the protocol described herein is suitable for hexyl-aminolevulinate-mediated photodynamic therapy and diagnosis when protoporphyrin IX is the active agent.

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 Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats

Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 348 ◽  
Author(s):  
Leticia Díez-Quijada ◽  
Concepción Medrano-Padial ◽  
María Llana-Ruiz-Cabello ◽  
Giorgiana M. Cătunescu ◽  
Rosario Moyano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Fatty Degeneration ◽  
Dna Strand Breaks ◽  
Histopathological Study ◽  
Strand Breaks ◽  
Dna Strand

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

scholarly journals Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

1986 ◽  
Vol 6 (11) ◽  
pp. 3831-3837 ◽  
Author(s):  
M Jayaram
Keyword(s):  
Mating Type ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Conversion Event ◽  
Yeast Mating ◽  
Mating Type Switching

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.

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.

In vitro and in vivo expression of protoporphyrin IX induced by lipophilic 5-aminolevulinic acid derivatives

2001 ◽  
Vol 27 (2) ◽  
pp. 114-120 ◽  
Author(s):  
Yoshiharu Ninomiya ◽  
Yoshiyasu Itoh ◽  
Shingo Tajima ◽  
Akira Ishibashi
Keyword(s):  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
In Vivo Expression
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