In vitro Effect of Harmine Alkaloid and Its N-Methyl Derivatives Against Toxoplasma gondii

Toxoplasmosis is one of the most prevalent and neglected zoonotic global diseases caused by Toxoplasma gondii. The current pharmacological treatments show clinical limitations, and therefore, the search for new drugs is an urgent need in order to eradicate this infection. Due to their intrinsic biological activities, β-carboline (βC) alkaloids might represent a good alternative that deserves further investigations. In this context, the in vitro anti-T. gondii activity of three βCs, harmine (1), 2-methyl-harminium (2), and 9-methyl-harmine (3), was evaluated herein. Briefly, the three alkaloids exerted direct effects on the parasite invasion and/or replication capability. Replication rates of intracellular treated tachyzoites were also affected in a dose-dependent manner, at noncytotoxic concentrations for host cells. Additionally, cell cycle analysis revealed that both methyl-derivatives 2 and 3 induce parasite arrest in S/M phases. Compound 3 showed the highest irreversible parasite growth inhibition, with a half maximal inhibitory concentration (IC50) value of 1.8 ± 0.2 μM and a selectivity index (SI) of 17.2 at 4 days post infection. Due to high replication rates, tachyzoites are frequently subjected to DNA double-strand breaks (DSBs). This highly toxic lesion triggers a series of DNA damage response reactions, starting with a kinase cascade that phosphorylates a large number of substrates, including the histone H2A.X to lead the early DSB marker γH2A.X. Western blot studies showed that basal expression of γH2A.X was reduced in the presence of 3. Interestingly, the typical increase in γH2A.X levels produced by camptothecin (CPT), a drug that generates DSB, was not observed when CPT was co-administered with 3. These findings suggest that 3 might disrupt Toxoplasma DNA damage response.

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

Parasites & Vectors ◽

10.1186/s13071-020-04324-7 ◽

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.

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

10.21203/rs.3.rs-35853/v1 ◽

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.

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The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

NAR Cancer ◽

10.1093/narcan/zcab002 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Luisa Statello ◽

Mohamad M Ali ◽

Silke Reischl ◽

Sagar Mahale ◽

Subazini Thankaswamy Kosalai ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cancer Biology ◽

Topoisomerase I ◽

Cell Cycle Checkpoints ◽

Topoisomerase 1 ◽

Damage Response ◽

Promote Cell Survival

Abstract Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1–cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

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PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2

Cell Death and Disease ◽

10.1038/s41419-020-2708-5 ◽

2020 ◽

Vol 11 (7) ◽

Author(s):

Nan Huang ◽

Chang Xu ◽

Liang Deng ◽

Xue Li ◽

Zhixuan Bian ◽

...

Keyword(s):

Dna Damage ◽

Histone Deacetylase ◽

Dna Damage Response ◽

De Novo ◽

Dna Damage Repair ◽

Histone Deacetylase 1 ◽

Damage Repair ◽

Damage Response

AbstractPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.

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Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

Journal of Biological Chemistry ◽

10.1074/jbc.c900032200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16066-16070 ◽

Cited By ~ 88

Author(s):

Navasona Krishnan ◽

Dae Gwin Jeong ◽

Suk-Kyeong Jung ◽

Seong Eon Ryu ◽

Andrew Xiao ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Mammalian Cells ◽

Protein Tyrosine Phosphatases ◽

Histone H2a ◽

Eyes Absent ◽

Damage Repair ◽

Damage Response

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.

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HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma

Oncotarget ◽

10.18632/oncotarget.5512 ◽

2015 ◽

Vol 6 (33) ◽

pp. 34979-34991 ◽

Cited By ~ 6

Author(s):

Yuezhen Xue ◽

Shen Yon Toh ◽

Pingping He ◽

Thimothy Lim ◽

Diana Lim ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cervical Carcinoma ◽

Precursor Lesions ◽

Damage Response ◽

Cellular Dna

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In Vitro Biological Characterization of DCUN1D5 in DNA Damage Response

Asian Pacific Journal of Cancer Prevention ◽

10.7314/apjcp.2012.13.8.4157 ◽

2012 ◽

Vol 13 (8) ◽

pp. 4157-4162 ◽

Cited By ~ 5

Author(s):

Wei Guo ◽

Guo-Jun Li ◽

Hong-Bo Xu ◽

Jie-Shi Xie ◽

Tai-Ping Shi ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Biological Characterization ◽

Damage Response

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ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

Journal of Nutrition ◽

10.1093/jn/nxz309 ◽

2019 ◽

Vol 150 (5) ◽

pp. 1022-1030 ◽

Cited By ~ 5

Author(s):

Dandan Xu ◽

Weiwei Dai ◽

Lydia Kutzler ◽

Holly A Lacko ◽

Leonard S Jefferson ◽

...

Keyword(s):

Amino Acids ◽

Dna Damage ◽

Amino Acid ◽

Protein Kinase ◽

Dna Damage Response ◽

Dependent Manner ◽

Mouse Embryo Fibroblasts ◽

Damage Response ◽

Mtorc1 Activation ◽

In Cells

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

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