scholarly journals Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

2018 ◽  
Author(s):  
Rachel J. Carter ◽  
Catherine M. Nickson ◽  
James M. Thompson ◽  
Andrzej Kacperek ◽  
Mark A. Hill ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Cellular Response ◽  
Cell Killing ◽  
Dna Lesions ◽  
Ionising Radiation ◽  
X Rays ◽  
High Let Radiation ◽  
High Let ◽  
Parp 1

AbstractPurposeIonising radiation, particular high linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) where two or more DNA lesions are induced in close proximity which contributes significantly to the cell killing effects. However knowledge of the enzymes and mechanisms involved in co-ordinating the recognition and processing of CDD in cellular DNA are currently lacking.Methods and MaterialsAn siRNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET -particles or protons, versus low-LET protons and x-rays, and cell survival monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed by using an enzyme modified neutral comet assay.ResultsDepletion of USP6 decreased cell survival specifically following high-LET α-particles and protons, but not by low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1). This phenotype was mimicked using the PARP inhibitor olaparib.ConclusionUSP6 controls cell survival in response to high-LET radiation by stabilising PARP-1 protein levels which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition in effective cancer cell killing.

scholarly journals USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation

2021 ◽  
Vol 11 ◽  
Author(s):  
Catherine M. Nickson ◽  
Maria Rita Fabbrizi ◽  
Rachel J. Carter ◽  
Jonathan R. Hughes ◽  
Andrzej Kacperek ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Cell Biology ◽  
Cellular Response ◽  
Biological Effects ◽  
Control Cell ◽  
X Rays ◽  
High Let Radiation ◽  
High Let ◽  
The Impact

Ionizing radiation (IR) principally acts through induction of DNA damage that promotes cell death, although the biological effects of IR are more broad ranging. In fact, the impact of IR of higher-linear energy transfer (LET) on cell biology is generally not well understood. Critically, therefore, the cellular enzymes and mechanisms responsible for enhancing cell survival following high-LET IR are unclear. To this effect, we have recently performed siRNA screening to identify deubiquitylating enzymes that control cell survival specifically in response to high-LET α-particles and protons, in comparison to low-LET X-rays and protons. From this screening, we have now thoroughly validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using small interfering RNA (siRNA), leads to significantly decreased survival of cells after high-LET radiation. We consequently investigated the mechanism through which this occurs, and demonstrate that an absence of USP9X has no impact on DNA damage repair post-irradiation nor on apoptosis, autophagy, or senescence. We discovered that USP9X is required to stabilize key proteins (CEP55 and CEP131) involved in centrosome and cilia formation and plays an important role in controlling pericentrin-rich foci, particularly in response to high-LET protons. This was also confirmed directly by demonstrating that depletion of CEP55/CEP131 led to both enhanced radiosensitivity of cells to high-LET protons and amplification of pericentrin-rich foci. Our evidence supports the importance of USP9X in maintaining centrosome function and biogenesis and which is crucial particularly in the cellular response to high-LET radiation.

scholarly journals Immunomodulatory Effects of Radiotherapy

2020 ◽  
Vol 21 (21) ◽  
pp. 8151
Author(s):  
Sharda Kumari ◽  
Shibani Mukherjee ◽  
Debapriya Sinha ◽  
Salim Abdisalaam ◽  
Sunil Krishnan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Carbon Particle ◽  
Dna Lesions ◽  
X Rays ◽  
Adaptive Immune ◽  
High Let Radiation ◽  
Different Types ◽  
High Let ◽  
Radiation Induced ◽  
Tumor Death

Radiation therapy (RT), an integral component of curative treatment for many malignancies, can be administered via an increasing array of techniques. In this review, we summarize the properties and application of different types of RT, specifically, conventional therapy with x-rays, stereotactic body RT, and proton and carbon particle therapies. We highlight how low-linear energy transfer (LET) radiation induces simple DNA lesions that are efficiently repaired by cells, whereas high-LET radiation causes complex DNA lesions that are difficult to repair and that ultimately enhance cancer cell killing. Additionally, we discuss the immunogenicity of radiation-induced tumor death, elucidate the molecular mechanisms by which radiation mounts innate and adaptive immune responses and explore strategies by which we can increase the efficacy of these mechanisms. Understanding the mechanisms by which RT modulates immune signaling and the key players involved in modulating the RT-mediated immune response will help to improve therapeutic efficacy and to identify novel immunomodulatory drugs that will benefit cancer patients undergoing targeted RT.

scholarly journals Cooperation of the Cockayne Syndrome Group B Protein and Poly(ADP-Ribose) Polymerase 1 in the Response to Oxidative Stress

2005 ◽  
Vol 25 (17) ◽  
pp. 7625-7636 ◽  
Author(s):  
Tina Thorslund ◽  
Cayetano von Kobbe ◽  
Jeanine A. Harrigan ◽  
Fred E. Indig ◽  
Mette Christiansen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Cellular Response ◽  
Genetic Disorder ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Group B ◽  
Parp 1

ABSTRACT Cockayne syndrome (CS) is a rare genetic disorder characterized as a segmental premature-aging syndrome. The CS group B (CSB) protein has previously been implicated in transcription-coupled repair, transcriptional elongation, and restoration of RNA synthesis after DNA damage. Recently, evidence for a role of CSB in base excision repair of oxidative DNA lesions has accumulated. In our search to understand the molecular function of CSB in this process, we identify a physical and functional interaction between CSB and poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a nuclear enzyme that protects the integrity of the genome by responding to oxidative DNA damage and facilitating DNA repair. PARP-1 binds to single-strand DNA breaks which activate the catalytic ability of PARP-1 to add polymers of ADP-ribose to various proteins. We find that CSB is present at sites of activated PARP-1 after oxidative stress, identify CSB as a new substrate of PARP-1, and demonstrate that poly(ADP-ribosyl)ation of CSB inhibits its DNA-dependent ATPase activity. Furthermore, we find that CSB-deficient cell lines are hypersensitive to inhibition of PARP. Our results implicate CSB in the PARP-1 poly(ADP-ribosyl)ation response after oxidative stress and thus suggest a novel role of CSB in the cellular response to oxidative damage.

scholarly journals Strong Shift to ATR-Dependent Regulation of the G2-Checkpoint after Exposure to High-LET Radiation

Life ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 560
Author(s):  
Veronika Mladenova ◽  
Emil Mladenov ◽  
Michael Scholz ◽  
Martin Stuschke ◽  
George Iliakis
Keyword(s):  
Chromosomal Abnormalities ◽  
Biological Effects ◽  
Cell Killing ◽  
Space Environment ◽  
Low Doses ◽  
X Rays ◽  
G2 Checkpoint ◽  
High Let Radiation ◽  
High Let ◽  
Α Particles

The utilization of high linear-energy-transfer (LET) ionizing radiation (IR) modalities is rapidly growing worldwide, causing excitement but also raising concerns, because our understanding of their biological effects is incomplete. Charged particles such as protons and heavy ions have increasing potential in cancer therapy, due to their advantageous physical properties over X-rays (photons), but are also present in the space environment, adding to the health risks of space missions. Therapy improvements and the protection of humans during space travel will benefit from a better understanding of the mechanisms underpinning the biological effects of high-LET IR. There is evidence that high-LET IR induces DNA double-strand breaks (DSBs) of increasing complexity, causing enhanced cell killing, owing, at least partly, to the frequent engagement of a low-fidelity DSB-repair pathway: alternative end-joining (alt-EJ), which is known to frequently induce severe structural chromosomal abnormalities (SCAs). Here, we evaluate the radiosensitivity of A549 lung adenocarcinoma cells to X-rays, α-particles and 56Fe ions, as well as of HCT116 colorectal cancer cells to X-rays and α-particles. We observe the expected increase in cell killing following high-LET irradiation that correlates with the increased formation of SCAs as detected by mFISH. Furthermore, we report that cells exposed to low doses of α-particles and 56Fe ions show an enhanced G2-checkpoint response which is mainly regulated by ATR, rather than the coordinated ATM/ATR-dependent regulation observed after exposure to low doses of X-rays. These observations advance our understanding of the mechanisms underpinning high-LET IR effects, and suggest the potential utility for ATR inhibitors in high-LET radiation therapy.

Comparison of clonogenic cell survival and DNA damage induced by 188Re and X-rays in rat thyroid cells

2017 ◽  
Vol 56 (01) ◽  
pp. 47-54
Author(s):  
Jana Arlt ◽  
Liane Oehme ◽  
Robert Freudenberg ◽  
Jörg Kotzerke ◽  
Roswitha Runge
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Pearson Correlation ◽  
Dna Lesions ◽  
X Rays ◽  
Dsb Repair ◽  
Thyroid Cells ◽  
X Ray ◽  
Rat Thyroid ◽  
Spearman Test

Summary Aim: Ionizing radiation produces DNA lesions among which DNA double strand breaks (DSB) are the most critical events. Radiation of various energy types might differ in their biological effectiveness. Here, we compared cell survival and DNA damage induced by 188Re and X-rays using YH2AX foci as a measure of DSB. The correlation between survival and residual foci was also analyzed. Methods: PCCl3 cells were irradiated with 200 kV X-rays (1.2 Gy/min) or 0.5-25 MBq/ml 188Re (1 h irradiation) achieving doses up to 10 Gy. By blocking of sodium iodide sym- porter (NIS) essentially extracellular activity could be guaranteed. Survival fractions (SF) were detected by colony forming assay. Initial and residual YH2AX foci (15 min and 24 h after irradiation) were assessed by immunos- taining. The relationship between SF and residual radiation induced YH2AX foci (RIF) was evaluated by Spearman and Pearson correlation tests. Results: We did not find significant differences between the survival curves in terms of the radiation quality. The D37 values were 4.6 Gy and 4.2 Gy for 188Re or X-ray, respectively. The initial foci numbers were in the same range for 188Re and X-ray, but higher levels of residual foci persisted after X-rays in comparison to 188Re (1 GyX-ray 6.5 ± 0.2; 1 GyRe-188 4.8 ± 0.2 RIF). Accordingly, for 188Re a higher extent of DSB repair was found. The Spearman test revealed a significant (p < 0.01) correlation between SF and residual RIF for both radiation modalities. Conclusion: No differences in terms of radiation were found for SF and initial foci. However, residual foci were lower for 188Re than for X-rays. A prediction of SF by residual foci should consider the properties of the radiation qualities that influence foci removal and DSB repair.

scholarly journals Clustered DNA Double-Strand Breaks: Biological Effects and Relevance to Cancer Radiotherapy

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 99 ◽  
Author(s):  
Jac A. Nickoloff ◽  
Neelam Sharma ◽  
Lynn Taylor
Keyword(s):  
Dna Damage ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
High Let Radiation ◽  
High Let ◽  
Radiation Induced ◽  
Clustered Dna Damage

Cells manage to survive, thrive, and divide with high accuracy despite the constant threat of DNA damage. Cells have evolved with several systems that efficiently repair spontaneous, isolated DNA lesions with a high degree of accuracy. Ionizing radiation and a few radiomimetic chemicals can produce clustered DNA damage comprising complex arrangements of single-strand damage and DNA double-strand breaks (DSBs). There is substantial evidence that clustered DNA damage is more mutagenic and cytotoxic than isolated damage. Radiation-induced clustered DNA damage has proven difficult to study because the spectrum of induced lesions is very complex, and lesions are randomly distributed throughout the genome. Nonetheless, it is fairly well-established that radiation-induced clustered DNA damage, including non-DSB and DSB clustered lesions, are poorly repaired or fail to repair, accounting for the greater mutagenic and cytotoxic effects of clustered lesions compared to isolated lesions. High linear energy transfer (LET) charged particle radiation is more cytotoxic per unit dose than low LET radiation because high LET radiation produces more clustered DNA damage. Studies with I-SceI nuclease demonstrate that nuclease-induced DSB clusters are also cytotoxic, indicating that this cytotoxicity is independent of radiogenic lesions, including single-strand lesions and chemically “dirty” DSB ends. The poor repair of clustered DSBs at least in part reflects inhibition of canonical NHEJ by short DNA fragments. This shifts repair toward HR and perhaps alternative NHEJ, and can result in chromothripsis-mediated genome instability or cell death. These principals are important for cancer treatment by low and high LET radiation.

Alpha-Particle Exposure Induces Mainly Unstable Complex Chromosome Aberrations which do not Contribute to Radiation-Associated Cytogenetic Risk

2021 ◽  
Author(s):  
C. Hartel ◽  
E. Nasonova ◽  
S. Ritter ◽  
T. Friedrich
Keyword(s):  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Mitotic Cell ◽  
Peripheral Blood Lymphocytes ◽  
Small Scale ◽  
X Rays ◽  
Carcinogenic Effect ◽  
High Let Radiation ◽  
High Let ◽  
Α Particles

The mechanism underlying the carcinogenic potential of α radiation is not fully understood, considering that cell inactivation (e.g., mitotic cell death) as a main consequence of exposure efficiently counteracts the spreading of heritable DNA damage. The aim of this study is to improve our understanding of the effectiveness of α particles in inducing different types of chromosomal aberrations, to determine the respective values of the relative biological effectiveness (RBE) and to interpret the results with respect to exposure risk. Human peripheral blood lymphocytes (PBLs) from a single donor were exposed ex vivo to doses of 0–6 Gy X rays or 0–2 Gy α particles. Cells were harvested at two different times after irradiation to account for the mitotic delay of heavily damaged cells, which is known to occur after exposure to high-LET radiation (including α particles). Analysis of the kinetics of cells reaching first or second (and higher) mitosis after irradiation and aberration data obtained by the multiplex fluorescence in situ hybridization (mFISH) technique are used to determine of the cytogenetic risk, i.e., the probability for transmissible aberrations in surviving lymphocytes. The analysis shows that the cytogenetic risk after α exposure is lower than after X rays. This indicates that the actually observed higher carcinogenic effect of α radiation is likely to stem from small scale mutations that are induced effectively by high-LET radiation but cannot be resolved by mFISH analysis.

scholarly journals MORC2 regulates DNA damage response through a PARP1-dependent pathway

2019 ◽  
Vol 47 (16) ◽  
pp. 8502-8520 ◽  
Author(s):  
Lin Zhang ◽  
Da-Qiang Li
Keyword(s):  
Dna Damage ◽  
Zinc Finger ◽  
Chromatin Remodeling ◽  
Dna Damage Response ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Genotoxic Stress ◽  
Underlying Mechanism ◽  
Dna Lesions ◽  
Damage Response

Abstract Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.

scholarly journals DNA Damage-Inducing Anticancer Therapies: From Global to Precision Damage

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2098 ◽  
Author(s):  
Thom G. A. Reuvers ◽  
Roland Kanaar ◽  
Julie Nonnekens
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Cellular Proliferation ◽  
Dna Damage Repair ◽  
Therapy Response ◽  
Dna Lesions ◽  
Response To Therapy ◽  
Novel Therapy ◽  
Damage Repair ◽  
And Function

DNA damage-inducing therapies are of tremendous value for cancer treatment and function by the direct or indirect formation of DNA lesions and subsequent inhibition of cellular proliferation. Of central importance in the cellular response to therapy-induced DNA damage is the DNA damage response (DDR), a protein network guiding both DNA damage repair and the induction of cancer-eradicating mechanisms such as apoptosis. A detailed understanding of DNA damage induction and the DDR has greatly improved our knowledge of the classical DNA damage-inducing therapies, radiotherapy and cytotoxic chemotherapy, and has paved the way for rational improvement of these treatments. Moreover, compounds targeting specific DDR proteins, selectively impairing DNA damage repair in cancer cells, form a promising novel therapy class that is now entering the clinic. In this review, we give an overview of the current state and ongoing developments, and discuss potential avenues for improvement for DNA damage-inducing therapies, with a central focus on the role of the DDR in therapy response, toxicity and resistance. Furthermore, we describe the relevance of using combination regimens containing DNA damage-inducing therapies and how they can be utilized to potentiate other anticancer strategies such as immunotherapy.

scholarly journals A theoretical cell-killing model to evaluate oxygen enhancement ratios at DNA damage and cell survival endpoints in radiation therapy

2020 ◽  
Vol 65 (9) ◽  
pp. 095006
Author(s):  
Yusuke Matsuya ◽  
Tatsuhiko Sato ◽  
Rui Nakamura ◽  
Shingo Naijo ◽  
Hiroyuki Date
Keyword(s):  
Dna Damage ◽  
Radiation Therapy ◽  
Cell Survival ◽  
Cell Killing ◽  
Survival Endpoints ◽  
Oxygen Enhancement
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