In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

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POGZ modulates the DNA damage response in a HP1-dependent manner

10.1101/2021.06.28.447216 ◽

2021 ◽

Author(s):

John Heath ◽

Estelle Simo Cheyou ◽

Steven Findlay ◽

Vincent Luo ◽

Edgar Pinedo Carpio ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Genome Stability ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

Humoral Immune ◽

Damage Response

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonic lethal and Pogz haplo-insufficiency (Pogz+/Δ) results in a developmental delay, a deficit in intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/Δ mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

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Nse2, a Component of the Smc5-6 Complex, Is a SUMO Ligase Required for the Response to DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.25.1.185-196.2005 ◽

2005 ◽

Vol 25 (1) ◽

pp. 185-196 ◽

Cited By ~ 146

Author(s):

Emily A. Andrews ◽

Jan Palecek ◽

John Sergeant ◽

Elaine Taylor ◽

Alan R. Lehmann ◽

...

Keyword(s):

Dna Damage ◽

Ring Finger ◽

Dependent Manner ◽

Dna Damaging Agents ◽

Sumo Ligase ◽

Essential Function ◽

Mutant Cells ◽

Smc Proteins

ABSTRACT The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M r complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex.

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Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Journal of Experimental Medicine ◽

10.1084/jem.20092437 ◽

2010 ◽

Vol 207 (5) ◽

pp. 983-997 ◽

Cited By ~ 73

Author(s):

Li Li ◽

Marie-Jo Halaby ◽

Anne Hakem ◽

Renato Cardoso ◽

Samah El Ghamrasni ◽

...

Keyword(s):

Dna Damage ◽

Class Switch Recombination ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Class Switch ◽

Physiological Processes ◽

Increased Sensitivity

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8.

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Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 induces DNA damage, cell death, and suppression of tumor growth in preclinical models of Ewing sarcoma

Oncogenesis ◽

10.1038/s41389-020-00264-0 ◽

2020 ◽

Vol 9 (9) ◽

Author(s):

Anna E. Gibson ◽

Choh Yeung ◽

Sameer H. Issaq ◽

Victor J. Collins ◽

Michael Gouzoulis ◽

...

Keyword(s):

Dna Damage ◽

Tumor Growth ◽

Ewing Sarcoma ◽

Mitochondrial Activity ◽

Dependent Manner ◽

Preclinical Models ◽

Single Digit ◽

Nicotinamide Phosphoribosyltransferase

Abstract NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.

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Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3277-3285.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3277-3285 ◽

Cited By ~ 44

Author(s):

Takahiro Naiki ◽

Tatsushi Wakayama ◽

Daisuke Nakada ◽

Kunihiro Matsumoto ◽

Katsunori Sugimoto

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Dna Damage Checkpoint ◽

Dependent Manner ◽

Wild Type ◽

Strand Breaks ◽

Dependent Mechanism ◽

In Cells

ABSTRACT Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Δ or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Δ mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.

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E2F3 Is a Mediator of DNA Damage-Induced Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.00938-09 ◽

2009 ◽

Vol 30 (2) ◽

pp. 524-536 ◽

Cited By ~ 40

Author(s):

Luis A. Martinez ◽

Elzbieta Goluszko ◽

Hui-Zi Chen ◽

Gustavo Leone ◽

Sean Post ◽

...

Keyword(s):

Dna Damage ◽

Phosphorylation Site ◽

Dependent Manner ◽

The Novel ◽

Checkpoint Kinases ◽

Damage Response ◽

E2f Transcription Factors ◽

Induced Apoptosis

ABSTRACT The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.

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Protective Effects of Extracts fromFructus rhodomyrtiagainst Oxidative DNA DamageIn VitroandIn Vivo

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/507407 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Yuebin Ke ◽

Xinyun Xu ◽

Shuang Wu ◽

Juan Huang ◽

Yijie Geng ◽

...

Keyword(s):

Dna Damage ◽

Oxidative Dna Damage ◽

Antioxidant Properties ◽

Dependent Manner ◽

Protective Effects ◽

Concentration Dependent Manner ◽

Spleen Lymphocytes ◽

Endogenous Antioxidant

Objective. To evaluate the potential protective effects of extracts fromFructus rhodomyrti(FR) against oxidative DNA damage using a cellular system and the antioxidant ability onpotassiumbromate- (KBrO3-) mediated oxidative stress in rats.Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3.Results. FR was shown to effectively protect against DNA damage induced by H2O2 in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats.Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent mannerin vitroandin vivo.

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Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

Protein and Peptide Letters ◽

10.2174/0929866526666190327130037 ◽

2019 ◽

Vol 26 (7) ◽

pp. 494-501 ◽

Cited By ~ 5

Author(s):

Sameer Suresh Bhagyawant ◽

Dakshita Tanaji Narvekar ◽

Neha Gupta ◽

Amita Bhadkaria ◽

Ajay Kumar Gautam ◽

...

Keyword(s):

Biological Effects ◽

Exchange Chromatography ◽

Health Concern ◽

Carbohydrate Binding ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ic50 Values ◽

Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

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A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666181207102037 ◽

2019 ◽

Vol 19 (3) ◽

pp. 365-374 ◽

Cited By ~ 1

Author(s):

Yang Liu ◽

Jingyin Zhang ◽

Shuyun Feng ◽

Tingli Zhao ◽

Zhengzheng Li ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Dna Damage ◽

Cyclin D ◽

Dna Relaxation ◽

Cycle Arrest ◽

H460 Cell ◽

H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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Protective effect of argan oil on DNA damage in vivo and in vitro

Biomarkers ◽

10.1080/1354750x.2021.1905068 ◽

2021 ◽

pp. 1-9

Author(s):

Habiba Bouchab ◽

Abbas Ishaq ◽

Riad EL Kebbaj ◽

Boubker Nasser ◽

Gabriele Saretzki

Keyword(s):

Dna Damage ◽

Protective Effect ◽

Argan Oil

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