Depletion of the 110-Kilodalton Isoform of Poly(ADP-Ribose) Glycohydrolase Increases Sensitivity to Genotoxic and Endotoxic Stress in Mice

ABSTRACT Poly(ADP-ribosylation) is rapidly stimulated in cells following DNA damage. This posttranslational modification is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Although the role of PARP-1 in response to DNA damage has been studied extensively, the function of PARG and the impact of poly(ADP-ribose) homeostasis in various cellular processes are largely unknown. Here we show that by gene targeting in embryonic stem cells and mice, we specifically deleted the 110-kDa PARG protein (PARG110) normally found in the nucleus and that depletion of PARG110 severely compromised the automodification of PARP-1 in vivo. PARG110-deficient mice were viable and fertile, but these mice were hypersensitive to alkylating agents and ionizing radiation. In addition, these mice were susceptible to streptozotocin-induced diabetes and endotoxic shock. These data indicate that PARG110 plays an important role in DNA damage responses and in pathological processes.

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Comprehensive analysis of yeast ESCRT-III composition in single ESCRT-III deletion mutants

Biochemical Journal ◽

10.1042/bcj20190141 ◽

2019 ◽

Vol 476 (14) ◽

pp. 2031-2046 ◽

Cited By ~ 2

Author(s):

Christian Heinzle ◽

Lara Mücke ◽

Thomas Brune ◽

Ralf Kölling

Keyword(s):

Family Members ◽

Cell Fractionation ◽

Endosomal Sorting ◽

Cellular Processes ◽

Associated Proteins ◽

Bona Fide ◽

Main Component ◽

The Impact

Abstract The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our findings are in line with the current concepts about ESCRT-III, but some findings are unexpected and call for adjustments to the model. In particular, our data suggest that the distinction between bona fide ESCRT-III components and ESCRT-III associated proteins is not justified. We detected a single complex containing all ESCRT-III members (except of Chm7) with Did2 as its main component. The classical core components were present in equimolar amounts. Our analysis of the impact of single deletions on the composition of ESCRT-III confirmed the central role of Snf7 for ESCRT-III assembly. For the other ESCRT-III family members predictions could be made about their role in ESCRT-III assembly. Furthermore, our cell fractionation points to a role of Vps20 at the endoplasmic reticulum.

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BIOL-11. THE ROLE OF ABERRANT EXPRESSION OF PRDM6 IN THE DEVELOPING CEREBELLUM AND IN GROUP 4 MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab090.018 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i5-i5

Author(s):

Christin Schmidt ◽

Annika Carlson ◽

William Weiss ◽

Bjoern Schwer

Keyword(s):

Stem Cells ◽

Embryonic Stem ◽

Genetic Alterations ◽

Cerebellar Development ◽

Aberrant Expression ◽

Group 4 ◽

Mycn Expression ◽

The Impact

Abstract Group 4 medulloblastoma is the most common medulloblastoma subgroup with an intermediate prognosis and a high incidence of metastasis and late-onset relapse cases. Despite several comprehensive genomic studies in medulloblastoma, Group 4 medulloblastomas lack a unifying oncogenic driver and treatment targets. This subgroup is characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and enhancer hijacking events. 17% of Group 4 medulloblastoma cases are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. PRDM6 amplified medulloblastoma cases show additional mutations in other chromatin regulators, such as KDM6A, KMT2C and KMT2D, ZMYM3, and high MYCN expression. In this project, we investigate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and the developing mouse cerebellum. We drive expression of PRDM6 in human iPSC-derived neuroepithelial stem cells (NESCs) with and without high MYCN expression to study its implications in tumorigenesis. To test for tumor growth in vivo and changes in tumor progression as a function of PRDM6 activity, NESCs are injected into the cerebellum of adult mice. In order to elucidate impact of PRDM6 activity during embryonic cerebellar development, we also introduce PRDM6 expression into mouse embryonic stem cells (ESCs) for analysis via a new, in vivo cerebellar blastocyst complementation model. The latter approach is designed to ablate and repopulate early granule neural precursor cells in the embryonal cerebellum with progenitors derived from injected PRDM6-ESCs and thus to recapitulate pre- and postnatal cerebellar development in vivo. Together, our studies aim to understand the role of PRDM6 during normal cerebellar development and tumorigenesis and advance the understanding of the genetic drivers for Group 4 medulloblastoma.

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Dynamic measurement of cytosolic pH and [NO3-] uncovers the role of the vacuolar transporter AtCLCa in the control of cytosolic pH

10.1101/716050 ◽

2019 ◽

Author(s):

Elsa Demes ◽

Laetitia Besse ◽

Béatrice Satiat-Jeunemaitre ◽

Sébastien Thomine ◽

Alexis De Angeli

Keyword(s):

Arabidopsis Thaliana ◽

Ion Homeostasis ◽

Ion Transporters ◽

Cytosolic Ph ◽

Major Function ◽

Cellular Processes ◽

Intracellular Compartments ◽

The Impact

AbstractIon transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile the understanding of their exact functions in the whole cell homeostasis is limited by the difficulty to monitor their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides an invaluable key to tackle this challenging issue. Here, we adapted the use of a dual biosensor using guard cells as experimental model to visualize the impact on the cytosol of anion transport from intracellular compartments. To image the activity of AtCLCa, a vacuolar NO3-/H+ exchanger regulating stomata aperture in Arabidopsis thaliana, we expressed a genetically encoded biosensor, ClopHensor allowing monitoring the dynamics of cytosolic anion concentration and pH. We first show that ClopHensor is not only a Cl- but also a NO3- sensor. We were then able to unravel and quantify the variations of NO3- and pH in the cytosol. Our data show that AtCLCa activity modifies cytosolic pH and NO3-, demonstrating that the transport activity of a vacuolar exchanger has a profound impact on cytosolic homeostasis. We propose that a major function of this endomembrane transporter is to adjust cytosolic conditions to cellular needs. This opens a novel perspective on the function of intracellular transporters of the CLC family in eukaryotes: not only controlling the intra organelle lumen but also actively modifying cytosolic conditions.SignificanceIntracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. The role of intracellular ion transporters is usually seen through an “intra organelle” lens, meanwhile their potential action on cytosolic ion homeostasis is still a black box. The case of a plant CLC is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the role of an Arabidopsis thaliana CLC, AtCLCa, in the modification of cytosolic pH. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.

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Leveraging the DNA damage and transcriptional-regulatory roles of PARP-1 as a means to improve prostate cancer therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.179 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 179-179

Author(s):

Matthew Joseph Schiewer ◽

Jamin Steffen ◽

Renee Tholey ◽

Jonathan Robert Brody ◽

John Pascal ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Dna Damage ◽

Cell Growth ◽

Target Gene ◽

Parp Inhibitors ◽

Transcriptional Regulatory ◽

The Impact ◽

Parp 1

179 Background: The first described roles for PARP-1 were in the repair of DNA damage and genomic maintenance, however, recent studies have identified PARP-1 as harboring critical context-dependent transcriptional regulatory functions. Our group recently discovered that PARP-1 enzymatic activity is a critical effector of androgen receptor (AR) function in models of prostatic adenocarcinoma (PCa), and is recruited to regulatory sites of select AR target genes. Pharmacological inhibition of PARP-1 enzymatic activity results in diminished AR and PARP-1 residency at AR target gene regulatory loci, reduced AR target gene expression (more than 50%), and reduced AR-driven, PCa-associated phenotypes, including castrate-resistant PCa (CRPC) AR function, tumor cell growth, and transition to CRPC. These data, in part, served as critical rationale for current clinical trials combining PARP inhibition and abiraterone for patients with metastatic CRPC NCT01576172 . Given the clinical importance of targeting AR function in PCa, preclinical studies were performed to assess the impact of leveraging the dual roles of PARP-1 as a means to improve therapy for advanced disease. Methods: In vitro and in vivo model systems were utilized to assess impact on AR and tumor growth. Multiple PARP inhibitors were used for additional studies. The impact of PARP-1 on gene expression was also assessed using unbiased analyses. PARP-1 mutants were also utilized to segregate the DNA damage and transcriptional regulatory roles of PARP-1. Results: Critically, PARP-1 inhibitors cooperated with castration to elicit an enhanced therapeutic response, and PARP-1 inhibitors were effective at suppressing CRPC growth in vivo. Multiple PARP inhibitors diminished AR chromatin occupancy, altered AR transcriptional output and reduction in PCa and CRPC cell growth. Conclusions: These data identify PARP-1 as a feasible therapeutic target for advanced prostate cancer. Multiple PARP inhibitors show pre-clinical efficacy in models of PCa and CRPC and merit consideration for clinical trial, and a concept will be presented.

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Laminin γ1 C-terminal Glu to Gln mutation induces early postimplantation lethality

Life Science Alliance ◽

10.26508/lsa.201800064 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800064 ◽

Cited By ~ 4

Author(s):

Daiji Kiyozumi ◽

Yukimasa Taniguchi ◽

Itsuko Nakano ◽

Junko Toga ◽

Emiko Yagi ◽

...

Keyword(s):

Animal Models ◽

Mouse Strain ◽

Basement Membranes ◽

Cellular Processes ◽

On Demand ◽

The Impact ◽

Laminin Binding

Laminin–integrin interactions regulate various adhesion-dependent cellular processes. γ1C-Glu, the Glu residue in the laminin γ1 chain C-terminal tail, is crucial for the binding of γ1-laminins to several integrin isoforms. Here, we investigated the impact of γ1C Glu to Gln mutation on γ1-laminin binding to all possible integrin partners in vitro, and found that the mutation specifically ablated binding to α3, α6, and α7 integrins. To examine the physiological significance of γ1C-Glu, we generated a knock-in allele, Lamc1EQ, in which the γ1C Glu to Gln mutation was introduced. Although Lamc1EQ/EQ homozygotes developed into blastocysts and deposited laminins in their basement membranes, they died just after implantation because of disordered extraembryonic development. Given the impact of the Lamc1EQ allele on embryonic development, we developed a knock-in mouse strain enabling on-demand introduction of the γ1C Glu to Gln mutation by the Cre-loxP system. The present study has revealed a crucial role of γ1C-Glu–mediated integrin binding in postimplantation development and provides useful animal models for investigating the physiological roles of laminin–integrin interactions in vivo.

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Essential Role of AKT-1/Protein Kinase Bα in PTEN-Controlled Tumorigenesis

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3842-3851.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3842-3851 ◽

Cited By ~ 99

Author(s):

Bangyan Stiles ◽

Valeriya Gilman ◽

Natalya Khanzenzon ◽

Ralf Lesche ◽

Annie Li ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Survival ◽

Essential Role ◽

Es Cells ◽

Familial Cancer ◽

Embryonic Stem ◽

Cellular Processes ◽

Downstream Effectors

ABSTRACT PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP3) accumulation, enhanced entry into S phase, and better cell survival. Since PIP3 controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten −/− ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten −/− ES cells. Deletion of Akt-1 partially reverses the aggressive growth of Pten −/− ES cells in vivo, suggesting that AKT-1 plays an essential role in PTEN-controlled tumorigenesis.

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Curcumin-containing Silver Nanoparticles prevent carbon tetrachlorideinduced hepatotoxicity in mice

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323666201211100830 ◽

2020 ◽

Vol 23 ◽

Author(s):

Hossam Ebaid ◽

Mohamed Habila ◽

Iftekhar Hassan ◽

Jameel Al-Tamimi ◽

Mohamed S. Omar ◽

...

Keyword(s):

Dna Damage ◽

Silver Nanoparticles ◽

Nuclear Dna ◽

Liquid Paraffin ◽

Tail Length ◽

Hepatic Tissue ◽

Tissue Destruction ◽

Group 2 ◽

The Impact

Background: Hepatotoxicity remains an important clinical challenge. Hepatotoxicity observed in response to toxins and hazardous chemicals may be alleviated by delivery of the curcumin in silver nanoparticles (AgNPs-curcumin). In this study, we examined the impact of AgNPs-curcumin in a mouse model of carbon tetrachloride (CCl4)-induced hepatic injury. Methods: Male C57BL/6 mice were divided into three groups (n=8 per group). Mice in group 1 were treated with vehicle control alone, while mice in Group 2 received a single intraperitoneal injection of 1 ml/kg CCl4 in liquid paraffin (1:1 v/v). Mice in group 3 were treated with 2.5 mg/kg AgNPs-curcumin twice per week for three weeks after the CCl4 challenge. Results: Administration of CCL4 resulted in oxidative dysregulation, including significant reductions in reduced glutathione and concomitant elevations in the level of malondialdehyde (MDA). CCL4 challenge also resulted in elevated levels of serum aspartate transaminase (AST) and alanine transaminase (ALT); these findings were associated with the destruction of hepatic tissues. Treatment with AgNPs-curcumin prevented oxidative imbalance, hepatic dysfunction, and tissue destruction. A comet assay revealed that CCl4 challenge resulted in significant DNA damage as documented by a 70% increase in nuclear DNA tail-length; treatment with AgNPs-curcumin inhibited the CCL4-mediated increase in nuclear DNA tail-length by 34%. Conclusion: Administration of AgNPs-curcumin resulted in significant antioxidant activity in vivo. This agent has the potential to prevent the hepatic tissue destruction and DNA damage that results from direct exposure to CCL4.

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The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

Nucleic Acids Research ◽

10.1093/nar/27.16.3276 ◽

1999 ◽

Vol 27 (16) ◽

pp. 3276-3282 ◽

Cited By ~ 65

Author(s):

P. P. H. Van Sloun ◽

J. G. Jansen ◽

G. Weeda ◽

L. H. F. Mullenders ◽

A. A. van Zeeland ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Embryonic Stem ◽

Genotoxic Effects ◽

Nucleotide Excision

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LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

Cell Death and Disease ◽

10.1038/s41419-021-03728-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

You-hong Wang ◽

Zhen Guo ◽

Liang An ◽

Yong Zhou ◽

Heng Xu ◽

...

Keyword(s):

Dna Damage ◽

Nasopharyngeal Cancer ◽

Noncoding Rnas ◽

Dependent Protein Kinase ◽

Damage Repair ◽

Dependent Protein ◽

Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

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Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22020501 ◽

2021 ◽

Vol 22 (2) ◽

pp. 501

Author(s):

Kateřina Skopalová ◽

Katarzyna Anna Radaszkiewicz ◽

Věra Kašpárková ◽

Jaroslav Stejskal ◽

Patrycja Bober ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Active Role ◽

Low Molecular Weight ◽

Conducting Materials ◽

The Impact ◽

Erk Kinase

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

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