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Sample records for poly-adp ribose polymerase

  1. Trapping Poly(ADP-Ribose) Polymerase.

    PubMed

    Shen, Yuqiao; Aoyagi-Scharber, Mika; Wang, Bing

    2015-06-01

    Recent findings indicate that a major mechanism by which poly(ADP-ribose) polymerase (PARP) inhibitors kill cancer cells is by trapping PARP1 and PARP2 to the sites of DNA damage. The PARP enzyme-inhibitor complex "locks" onto damaged DNA and prevents DNA repair, replication, and transcription, leading to cell death. Several clinical-stage PARP inhibitors, including veliparib, rucaparib, olaparib, niraparib, and talazoparib, have been evaluated for their PARP-trapping activity. Although they display similar capacity to inhibit PARP catalytic activity, their relative abilities to trap PARP differ by several orders of magnitude, with the ability to trap PARP closely correlating with each drug's ability to kill cancer cells. In this article, we review the available data on molecular interactions between these clinical-stage PARP inhibitors and PARP proteins, and discuss how their biologic differences might be explained by the trapping mechanism. We also discuss how to use the PARP-trapping mechanism to guide the development of PARP inhibitors as a new class of cancer therapy, both for single-agent and combination treatments. PMID:25758918

  2. Purification of recombinant poly(ADP-ribose) polymerases.

    PubMed

    Amé, Jean-Christophe; Kalisch, Thomas; Dantzer, Françoise; Schreiber, Valérie

    2011-01-01

    The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture. PMID:21870259

  3. Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

    PubMed Central

    Cantó, Carles; Sauve, Anthony A.; Bai, Peter

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

  4. Inhibiting poly(ADP-ribose) polymerase: a potential therapy against oligodendrocyte death

    PubMed Central

    Veto, Sara; Acs, Peter; Bauer, Jan; Lassmann, Hans; Berente, Zoltan; Setalo, Gyorgy; Borgulya, Gabor; Sumegi, Balazs; Komoly, Samuel; Gallyas, Ferenc; Illes, Zsolt

    2010-01-01

    Oligodendrocyte loss and demyelination are major pathological hallmarks of multiple sclerosis. In pattern III lesions, inflammation is minor in the early stages, and oligodendrocyte apoptosis prevails, which appears to be mediated at least in part through mitochondrial injury. Here, we demonstrate poly(ADP-ribose) polymerase activation and apoptosis inducing factor nuclear translocation within apoptotic oligodendrocytes in such multiple sclerosis lesions. The same morphological and molecular pathology was observed in an experimental model of primary demyelination, induced by the mitochondrial toxin cuprizone. Inhibition of poly(ADP-ribose) polymerase in this model attenuated oligodendrocyte depletion and decreased demyelination. Poly(ADP-ribose) polymerase inhibition suppressed c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation, increased the activation of the cytoprotective phosphatidylinositol-3 kinase-Akt pathway and prevented caspase-independent apoptosis inducing factor-mediated apoptosis. Our data indicate that poly(ADP-ribose) polymerase activation plays a crucial role in the pathogenesis of pattern III multiple sclerosis lesions. Since poly(ADP-ribose) polymerase inhibition was also effective in the inflammatory model of multiple sclerosis, it may target all subtypes of multiple sclerosis, either by preventing oligodendrocyte death or attenuating inflammation. PMID:20157013

  5. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity. PMID:23831359

  6. Increased poly(ADP-ribose)polymerase activity in cells infected by human immunodeficiency virus type-1.

    PubMed

    Furlini, G; Re, M C; La Placa, M

    1991-04-01

    Poly(ADP-ribose)polymerase is a chromatin-bound enzyme which is activated by free DNA ends and is therefore stimulated by a variety of DNA-damaging agents. The enzyme transfers the ADP moiety of NAD to nuclear proteins to create protein-bound ADP-ribose polymers. Under conditions favouring an accelerated poly(ADP-ribose) polymer formation, the enzyme may exhaust cellular NAD pools. At the same time, or shortly thereafter ATP levels drop and cell viability eventually declines. As a series of chemical and physical agents which may play a role in activating latent HIV-1 infection or favouring HIV-1 replication, have a DNA-damaging activity, we investigated the behaviour of poly(ADP-ribose)polymerase activity in various types of HIV-1-infected cells. The results obtained show that HIV-1-infected cells to possess an increased poly(ADP-ribosol)ating activity together with an accentuated fragmentation of cellular DNA which are associated with the time course of HIV-1 replication. These data give circumstantial support to the hypothesis that a NAD-depdendent cellular suicide response to DNA damage, could play a role in the death of HIV-1 infected cells. In this respect, the impared immunocompetence of HIV-1-infected patients could bear some resemblance to immune attribution that sometimes accompanies some inborn errors affecting DNA precursor metabolism and DNA integrity. PMID:1906973

  7. Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine

    PubMed Central

    Nilov, D. K.; Tararov, V. I.; Kulikov, A. V.; Zakharenko, A. L.; Gushchina, I. V.; Mikhailov, S. N.; Lavrik, O. I.; Švedas, V. K.

    2016-01-01

    The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 μM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment. PMID:27437145

  8. Structural analysis of poly(ADP-ribose)polymerase in higher and lower eukaryotes.

    PubMed

    Scovassi, A I; Izzo, R; Franchi, E; Bertazzoni, U

    1986-08-15

    A phylogenetic survey for the poly(ADP-ribose)polymerase has been conducted by analyzing enzyme activity in various organisms and determining the structure of the catalytic peptides by renaturation of functional activities of the enzyme in situ after electrophoresis in denaturing conditions (activity gel). The enzyme is widely distributed in cells from all different classes of vertebrates, from arthropods, mollusks and plant cells but could not be detected in echinoderms, nematodes, platyhelminths, thallophytes (including yeast) and bacteria. The presence on activity gels of a catalytic peptide with Mr = 115,000-120,000 was demonstrated in vertebrates, arthropods and mollusks but no activity bands were recovered in many lower eukaryotes, in plant cells and bacteria. By using an immunological procedure that used an antiserum against homogeneous calf thymus poly(ADP-ribose) polymerase, common immunoreactive peptides were visualized in mammals, avians, reptiles, amphibians and fishes, while lacking in non-vertebrate organisms. Our results indicate that the structure of poly(ADP-ribose) polymerase is conserved down to the mollusks suggesting its important role for DNA metabolism of multicellular organisms. PMID:3091369

  9. Does inhibition of poly(ADP-ribose) polymerase prevent energy overconsumption under microgravity?

    NASA Astrophysics Data System (ADS)

    Dobrota, C.; Piso, M. I.; Keul, A.

    When plants are exposed to a stress signal they expend a lot of energy and exhibit enhanced respiration rates This is partially due to a breakdown in the NAD pool caused by the enhanced activity PARP which uses NAD as a substrate to synthesize polymers of ADP-ribose Stress-induced depletion of NAD results in a similar depletion of energy since ATP molecules are required to resynthesize the depleted NAD It seems that plants with lowered poly ADP ribosyl ation activity appear tolerant to multiple stresses Inhibiting PARP activity prevents energy overconsumption under stress allowing normal mitochondrial respiration We intend to study if the microgravity is perceived by plants as a stress factor and if experimental inhibition of poly ADP-ribose polymerase may improve the energetic level of the cells References DeBlock M Verduyn C De Brouwer D and Cornelissen M 2005 Poly ADP-ribose polymerase in plants affects energy homeostasis cell death and stress tolerance The Plant Journal 41 95--106 Huang S Greenway H Colmerm T D and Millar A H 2005 Protein synthesis by rice coleoptiles during prolonged anoxia Implications for glycolysis growth and energy utilization Annals of Botany 96 703--715 Mittler R Vanderauwera S Gollery M and Van Breusegem F 2005 Reactive oxygen gene network of plants Trends in Plant Science 9 10 490-498

  10. Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

    PubMed Central

    Banasik, Marek; Stedeford, Todd; Strosznajder, Robert P; Takehashi, Masanori; Tanaka, Seigo; Ueda, Kunihiro

    2011-01-01

    Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of (β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation,and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl) ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4. PMID:21395487

  11. Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

    PubMed Central

    Hu, Biao; Wu, Zhe; Hergert, Polla; Henke, Craig A.; Bitterman, Peter B.; Phan, Sem H.

    2014-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with an expression plasmid activated expression of the α-SMA gene (Acta2), a marker of myofibroblast differentiation in lung fibroblasts. Suppression of PARP-1 activity or gene expression with PARP-1 inhibitors or siRNA, respectively, had the opposite effect on these cells. PARP-1–deficient cells also had reduced α-SMA gene expression. DNA pyrosequencing identified hypermethylated regions of the α-SMA gene in PARP-1–deficient cells, relative to wild-type cells. Interestingly, and of potential relevance to human idiopathic pulmonary fibrosis, PARP activity in lung fibroblasts isolated from idiopathic pulmonary fibrosis patients was significantly higher than that in cells isolated from control subjects. Furthermore, PARP-1–deficient mice exhibited reduced pulmonary fibrosis in response to bleomycin-induced lung injury, relative to wild-type controls. These results suggest that PARylation is important for myofibroblast differentiation and the pathogenesis of pulmonary fibrosis. PMID:23260200

  12. Regulation of myofibroblast differentiation by poly(ADP-ribose) polymerase 1.

    PubMed

    Hu, Biao; Wu, Zhe; Hergert, Polla; Henke, Craig A; Bitterman, Peter B; Phan, Sem H

    2013-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with an expression plasmid activated expression of the α-SMA gene (Acta2), a marker of myofibroblast differentiation in lung fibroblasts. Suppression of PARP-1 activity or gene expression with PARP-1 inhibitors or siRNA, respectively, had the opposite effect on these cells. PARP-1-deficient cells also had reduced α-SMA gene expression. DNA pyrosequencing identified hypermethylated regions of the α-SMA gene in PARP-1-deficient cells, relative to wild-type cells. Interestingly, and of potential relevance to human idiopathic pulmonary fibrosis, PARP activity in lung fibroblasts isolated from idiopathic pulmonary fibrosis patients was significantly higher than that in cells isolated from control subjects. Furthermore, PARP-1-deficient mice exhibited reduced pulmonary fibrosis in response to bleomycin-induced lung injury, relative to wild-type controls. These results suggest that PARylation is important for myofibroblast differentiation and the pathogenesis of pulmonary fibrosis. PMID:23260200

  13. Family-wide analysis of poly(ADP-ribose) polymerase activity

    PubMed Central

    Uchima, Lilen; Rood, Jenny; Zaja, Roko; Hay, Ronald T.; Ahel, Ivan; Chang, Paul

    2014-01-01

    The poly(ADP-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as substrate. Based on the composition of three NAD+ coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino acid targets. In addition, we identify cysteine as a novel amino acid target for ADP-ribosylation on PARPs. PMID:25043379

  14. Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark

    PubMed Central

    Lodhi, Niraj; Kossenkov, Andrew V.; Tulin, Alexei V.

    2014-01-01

    Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent. Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis. Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed. PMID:24861619

  15. PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres

    SciTech Connect

    Liu, Yie; Wu, Jun; Schreiber, Valerie; Dunlap, John; Dantzer, Francoise; Wang, Yisong

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  16. PARP1 is a TRF2-associated poly(ADP-ribose) polymerase and protects eroded telomeres

    SciTech Connect

    Gomez, Marla V; Wu, Jun; Wang, Yisong; Liu, Yie

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  17. Biology of Poly(ADP-Ribose) Polymerases: The Factotums of Cell Maintenance.

    PubMed

    Bai, Peter

    2015-06-18

    The protein family of poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin-type ADP-ribose transferases (ARTDs) are multidomain proteins originally identified as DNA repair factors. There are 17 PARP enzymes in humans, and it is now evident that PARPs undertake more tasks than DNA repair. The aim of this review is to give a comprehensive view of the biological roles of the PARP family starting from the simplest biochemical reactions to complex regulatory circuits. Special attention will be laid on discussing linkage of PARP enzymes with tumor biology, oxidative stress, inflammatory, and metabolic diseases. A better understanding of PARP-mediated processes and pathologies may help in identifying new pathways and, by these, new targets to combat diseases that affect large populations and seriously shorten life expectancy and the quality of life, such as cancer, metabolic, or inflammatory diseases. PMID:26091343

  18. Poly(ADP-ribose) polymerase 1 is a novel target to promote axonal regeneration

    PubMed Central

    Brochier, Camille; Jones, James I.; Willis, Dianna E.; Langley, Brett

    2015-01-01

    Therapeutic options for the restoration of neurological functions after acute axonal injury are severely limited. In addition to limiting neuronal loss, effective treatments face the challenge of restoring axonal growth within an injury environment where inhibitory molecules from damaged myelin and activated astrocytes act as molecular and physical barriers. Overcoming these barriers to permit axon growth is critical for the development of any repair strategy in the central nervous system. Here, we identify poly(ADP-ribose) polymerase 1 (PARP1) as a previously unidentified and critical mediator of multiple growth-inhibitory signals. We show that exposure of neurons to growth-limiting molecules—such as myelin-derived Nogo and myelin-associated glycoprotein—or reactive astrocyte-produced chondroitin sulfate proteoglycans activates PARP1, resulting in the accumulation of poly(ADP-ribose) in the cell body and axon and limited axonal growth. Accordingly, we find that pharmacological inhibition or genetic loss of PARP1 markedly facilitates axon regeneration over nonpermissive substrates. Together, our findings provide critical insights into the molecular mechanisms of axon growth inhibition and identify PARP1 as an effective target to promote axon regeneration. PMID:26598704

  19. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

    PubMed

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A; Mangerich, Aswin; Croteau, Deborah L; Bohr, Vilhelm A

    2015-12-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  20. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

    PubMed Central

    Khadka, Prabhat; Hsu, Joseph K.; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A.; Mangerich, Aswin; Croteau, Deborah L.

    2015-01-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  1. Poly(ADP-ribose) polymerase regulates glycolytic activity in kidney proximal tubule epithelial cells

    PubMed Central

    Song, Hana; Yoon, Sang Pil

    2016-01-01

    After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB–treated group and 36.7% decrease in 1 mM 3-AB–treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells. PMID:27382509

  2. PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses

    PubMed Central

    Song, Junqi; Keppler, Brian D.; Wise, Robert R.; Bent, Andrew F.

    2015-01-01

    Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation. PMID:25950582

  3. PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses.

    PubMed

    Song, Junqi; Keppler, Brian D; Wise, Robert R; Bent, Andrew F

    2015-05-01

    Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation. PMID:25950582

  4. Curcumin enhances poly(ADP-ribose) polymerase inhibitor sensitivity to chemotherapy in breast cancer cells.

    PubMed

    Choi, Young Eun; Park, Eunmi

    2015-12-01

    Poly(ADP-ribose) polymerase (PARP) inhibitor has shown promising responses in homologous recombination (HR) repair-deficient cancer cells. More specifically, targeting HR pathway in combination with PARP inhibitor has been an effective chemotherapy strategy by so far. Curcumin has been recognized as anticancer agents for several types of cancers. Here, we demonstrate that curcumin inhibits a critical step in HR pathway, Rad51 foci formation, and accumulates γ-H2AX levels in MDA-MB-231 breast cancer cells. Curcumin also directly reduces HR and induces cell death with cotreatment of PARP inhibitor in MDA-MB-231 breast cancer cells. Moreover, curcumin, when combined with ABT-888, could effectively delayed breast tumor formation in vivo. Our study indicates that cotreatment of curcumin and PARP inhibitor might be useful for the combination chemotherapy for aggressive breast cancer treatment as a natural bioactive compound. PMID:26350251

  5. Poly(ADP-ribose) polymerase-1 and apoptosis inducing factor in neurotoxicity.

    PubMed

    Yu, Seong-Woon; Wang, Hongmin; Dawson, Ted M; Dawson, Valina L

    2003-12-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is the guardian of the genome acting as a sentinel for genomic damage. However, PARP-1 is also mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. The biochemistry underlying PARP-1-mediated cell death has remained elusive, although NAD(+) consumption and energy failure have been thought to be one of the possible molecular mechanisms. Recent observations link PARP-1 activation with translocation of apoptosis-inducing factor (AIF) to the nucleus and indicate that AIF is an essential downstream effector of PARP-1-mediated cell death. PARP-1 activation signals AIF release from the mitochondria, resulting in a novel, caspase-independent pathway of programmed cell death. These recent findings suggest that AIF maybe a target for development of future therapeutic treatment for many neurological disorders involving excitotoxicity. PMID:14678748

  6. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.

    PubMed

    Yu, Seong-Woon; Wang, Hongmin; Poitras, Marc F; Coombs, Carmen; Bowers, William J; Federoff, Howard J; Poirier, Guy G; Dawson, Ted M; Dawson, Valina L

    2002-07-12

    Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death. PMID:12114629

  7. Poly(ADP)-Ribose Polymerase-1 Inhibitors as a Potential Treatment for Cocaine Addiction.

    PubMed

    Scobie, Kimberly N

    2015-01-01

    As of 2008, according to the National Survey on Drug Use and Health, nearly 1.4 million Americans met the Diagnostic and Statistical Manual of Mental Disorders criteria for dependence or abuse of cocaine (in any form) in the past 12 months. However, there are no treatments for cocaine use disorders approved by the Federal Drug Administration (FDA). Alterations in gene regulation contribute significantly to the changes that occur in the brain, both structurally and functionally, and the resultant addictive phenotype that occurs with chronic exposure to drugs of abuse. The Emerging Targets of Cocaine Use Disorders meeting sought to explore novel targets for the treatment of stimulant use disorder. The evidence for a role of one novel target, Poly(ADP)-ribose polymerase-1 (PARP-1), was presented at the meeting and will be summarized in this review. PMID:26022260

  8. Poly (ADP-Ribose) Polymerase Inhibitors: Recent Advances and Future Development

    PubMed Central

    Scott, Clare L.; Swisher, Elizabeth M.; Kaufmann, Scott H.

    2015-01-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising activity in epithelial ovarian cancers, especially relapsed platinum-sensitive high-grade serous disease. Consistent with preclinical studies, ovarian cancers and a number of other solid tumor types occurring in patients with deleterious germline mutations in BRCA1 or BRCA2 seem to be particularly sensitive. However, it is also becoming clear that germline BRCA1/2 mutations are neither necessary nor sufficient for patients to derive benefit from PARP inhibitors. We provide an update on PARP inhibitor clinical development, describe recent advances in our understanding of PARP inhibitor mechanism of action, and discuss current issues in the development of these agents. PMID:25779564

  9. Oxidative-Nitrosative Stress and Poly(ADP-Ribose) Polymerase (PARP) Activation in Experimental Diabetic Neuropathy

    PubMed Central

    Obrosova, Irina G.; Drel, Viktor R.; Pacher, Pal; Ilnytska, Olga; Wang, Zhong Q.; Stevens, Martin J.; Yorek, Mark A.

    2008-01-01

    Poly(ADP-ribose) polymerase (PARP) activation, an important factor in the pathogenesis of diabetes complications, is considered a downstream effector of oxidative-nitrosative stress. However, some recent findings suggest that it is not necessarily the case and that PARP activation may precede and contribute to free radical and oxidant-induced injury. This study evaluated the effect of PARP inhibition on oxidative-nitrosative stress in diabetic peripheral nerve, vasa nervorum, aorta, and high glucose–exposed human Schwann cells. In vivo experiments were performed in control rats and streptozocin (STZ)-induced diabetic rats treated with and without the PARP inhibitor 3-aminobenzamide (ABA) (30 mg · kg−1 · day−1 i.p. for 2 weeks after 2 weeks of untreated diabetes). Human Schwann cells (HSC) (passages 7–10; ScienCell Research Labs) were cultured in 5.5 or 30 mmol/l glucose with and without 5 mmol/l ABA. Diabetes-induced increase in peripheral nerve nitrotyrosine immunoreactivity, epineurial vessel superoxide and nitrotyrosine immunoreactivities, and aortic superoxide production was reduced by ABA. PARP-1 (Western blot analysis) was abundantly expressed in HSC, and its expression was not affected by high glucose or ABA treatment. High-glucose–induced superoxide production and overexpression of nitrosylated and poly(ADP-ribosyl)ated protein, chemically reduced amino acid-(4)-hydroxynonenal adducts, and inducible nitric oxide synthase were decreased by ABA. We concluded that PARP activation contributes to superoxide anion radical and peroxynitrite formation in peripheral nerve, vasa nervorum, and aorta of STZ-induced diabetic rats and high-glucose–exposed HSC. The relations between oxidative-nitrosative stress and PARP activation in diabetes are bi-rather than unidirectional, and PARP activation cannot only result from but also lead to free radical and oxidant generation. PMID:16306359

  10. Poly(ADP-ribose) polymerase-1 modulates Nrf2-dependent transcription.

    PubMed

    Wu, Tongde; Wang, Xiao-Jun; Tian, Wang; Jaramillo, Melba C; Lau, Alexandria; Zhang, Donna D

    2014-02-01

    The basic leucine zipper transcription factor Nrf2 has emerged as a master regulator of intracellular redox homeostasis by controlling the expression of a battery of redox-balancing antioxidants and phase II detoxification enzymes. Under oxidative stress conditions, Nrf2 is induced at the protein level through redox-sensitive modifications on critical cysteine residues in Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation. Poly(ADP-ribose) polymerase-1 (PARP-1) is historically known to function in DNA damage detection and repair; however, recently PARP-1 has been shown to play an important role in other biochemical activities, such as DNA methylation and imprinting, insulator activity, chromosome organization, and transcriptional regulation. The exact role of PARP-1 in transcription modulation and the underlying mechanisms remain poorly defined. In this study, we report that PARP-1 forms complexes with the antioxidant response element (ARE) within the promoter region of Nrf2 target genes and upregulates the transcriptional activity of Nrf2. Interestingly, PARP-1 neither physically interacts with Nrf2 nor promotes the expression of Nrf2. In addition, PARP-1 does not target Nrf2 for poly(ADP-ribosyl)ation. Instead, PARP-1 interacts directly with small Maf proteins and the ARE of Nrf2 target genes, which augments ARE-specific DNA-binding of Nrf2 and enhances the transcription of Nrf2 target genes. Collectively, these results suggest that PARP-1 serves as a transcriptional coactivator, upregulating the transcriptional activity of Nrf2 by enhancing the interaction among Nrf2, MafG, and the ARE. PMID:24140708

  11. Poly(ADP-Ribose) Polymerases: New Players in the Pathogenesis of Exocrine Pancreatic Diseases.

    PubMed

    Martínez-Bosch, Neus; Fernández-Zapico, Martin E; Navarro, Pilar; Yélamos, José

    2016-02-01

    The poly(ADP-ribose) polymerase (PARP) enzymes were initially characterized as sensors of DNA breaks but are now known to play key roles not only in the DNA damage response but also in regulating numerous molecular processes, such as gene transcription. Furthermore, these polymerases have emerged as key players in the pathogenesis of multiple diseases, providing promising therapeutic targets for pathologies such as cardiovascular disorders, neurodegenerative diseases, and cancer. In recent years, PARPs have been implicated in the pathogenesis of pancreatitis and pancreatic cancer, and PARP inhibition has been proposed as a valuable strategy for treating these two important gastrointestinal tract disorders. For instance, in preclinical mouse models, pancreatitis was significantly attenuated after genetic or pharmacological PARP inactivation, and several clinical trials have demonstrated promising responses to PARP inhibitors in pancreatic cancer patients. In this review, we summarize the current understanding of PARP functions in these two dismal pathologies and discuss the next steps necessary to determine whether PARP inhibitors will finally make the difference in treating pancreatitis and pancreatic cancer successfully. PMID:26687988

  12. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    SciTech Connect

    Burgman, P.; Konings, A.W. )

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.

  13. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells.

    PubMed

    Burgman, P; Konings, A W

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms. PMID:2502817

  14. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis

    PubMed Central

    Andrabi, Shaida A.; Umanah, George K. E.; Chang, Calvin; Stevens, Daniel A.; Karuppagounder, Senthilkumar S.; Gagné, Jean-Philippe; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2014-01-01

    Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD+ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD+ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD+ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD+ depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD+ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase. PMID:24987120

  15. Targeting BRCA1 localization to augment breast tumor sensitivity to poly(ADP-ribose) polymerase inhibition

    PubMed Central

    Yang, Eddy S.; Nowsheen, Somaira; Rahman, Mohammad A.; Cook, Rebecca S.; Xia, Fen

    2013-01-01

    Poly(ADP-ribose) polymerase inhibitors have gained recent attention due to their highly selective killing of BRCA1/2 mutated and DNA double strand break (DSB) repair deficient tumors. Unfortunately, the majority of sporadic breast cancers carry wild-type BRCA1/2 and are proficient in DSB repair. We and others have shown that BRCA1 is a nuclear/cytoplasm shuttling protein which is transiently exported from the nucleus to the cytosol upon various stimuli. Thus, we hypothesized that depletion of nuclear BRCA1 would compromise DSB repair and subsequently render sporadic tumors susceptible to PARP inhibition. Indeed, in human sporadic breast cancer cells with functional BRCA1 and proficient DSB repair, a transient nuclear depletion of BRCA1 and subsequent HR repair deficit was induced with either truncated BRCA1 or irradiation. This rendered these human sporadic breast cancer cells susceptible to PARP inhibition. These observations were confirmed genetically using mislocated BRCA1 mutants as well as in vivo in mice bearing breast tumor xenografts. These data support the potential strategy of targeting BRCA1 location to convert BRCA1-proficient sporadic tumors to be susceptible to the synthetic lethal combination with PARP inhibitors. PMID:22962264

  16. Activation of poly(ADP-ribose) polymerase by sulfur mustard in HeLa cell cultures

    SciTech Connect

    Clark, O.E.; Smith, W.J.

    1993-05-13

    Poly(ADP-ribose) polymerase (PADPRP) E.C.2.4.2.30 has been proposed to play a key role in the NAD+ depletion following alkylation of DNA in sulfur mustard (HD) exposures. Papirmeister et al. (Fundam Appl Toxicol 5:Sl34, 1985) hypothesized that activation of PADPRP was central to the subsequent depletion of NAD+ and activation of proteolytic enzymes leading to vesication. NAD+ depletion following HD exposure has been previously documented and the results have been used to infer the effect of HD exposure on PADPRP. The present study was undertaken to demonstrate the direct effect of HD on PADPRP activity. HeLa cells culture were used as the model system. At 10 microns HD PADPRP activity was increased above the levels of controls in the first hour. The activity peaked at 4 hrs and by 6 hrs had returned to control levels. The 24-hour level of PADPRP activity was again elevated above the controls. The 100 microns HD exposures had maximal enzymatic response in HeLa cells within the first hour. The level had decreased 40% from the maximum by the second hour reaching a plateau at 30% of the maximum response after 4 hrs. Cells exposed to 100 microns HD showed enzyme levels at or below those seen with the 10 microns dose after 24 hours. The doses of HD used did not decrease viability as measured by trypan blue dye exclusion within 24 hr.

  17. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    PubMed

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  18. Poly (ADP-ribose) polymerases inhibitor, Zj6413, as a potential therapeutic agent against breast cancer.

    PubMed

    Zhou, Qin; Ji, Ming; Zhou, Jie; Jin, Jing; Xue, Nina; Chen, Ju; Xu, Bailing; Chen, Xiaoguang

    2016-05-01

    Poly (ADP-ribose) polymerases (PARPs) facilitate repairing of cancer cell DNA damage as a mean to promote cancer proliferation and metastasis. Inhibitors of PARPs which interfering DNA repair, in context of defects in other DNA repair mechanisms, can thus be potentially exploited to inhibit or even kill cancer cells. However, nondiscriminatory inhibition of PARPs, such as PARP2, may lead to undesired consequences. Here, we demonstrated the design and development of the Zj6413 as a potent and selective PARP1 catalytic inhibitor. It trapped PARP1/2 at damaged sites of DNA. As expected, the Zj6413 showed notable anti-tumor activity against breast cancer gene (BRCA) deficient triple negative breast cancers (TNBCs). Zj6413 treated breast cancers (BCs) showed an elevated level of DNA damage evidenced by the accumulation of γ-H2AX foci and DNA damaged related proteins. Zj6413 also induced G2/M arrest and cell death in the MX-1, MDA-MB-453 BC cells, exerted chemo-sensitizing effect on BRCA proficient cancer cells and potentiated Temozolomide (TMZ)'s cytotoxicity in MX-1 xenograft tumors mice. In conclusion, our study provided evidence that a new PARP inhibitor strongly inhibited the catalytic activity of PARPs, trapped them on nicked DNA and damaged the cancer cells, eventually inhibiting the growth of breast tumor cells in vitro and in vivo. PMID:26920250

  19. Poly(ADP-ribose)polymerase activity controls plant growth by promoting leaf cell number.

    PubMed

    Schulz, Philipp; Jansseune, Karel; Degenkolbe, Thomas; Méret, Michaël; Claeys, Hannes; Skirycz, Aleksandra; Teige, Markus; Willmitzer, Lothar; Hannah, Matthew A

    2014-01-01

    A changing global environment, rising population and increasing demand for biofuels are challenging agriculture and creating a need for technologies to increase biomass production. Here we demonstrate that the inhibition of poly (ADP-ribose) polymerase activity is a promising technology to achieve this under non-stress conditions. Furthermore, we investigate the basis of this growth enhancement via leaf series and kinematic cell analysis as well as single leaf transcriptomics and plant metabolomics under non-stress conditions. These data indicate a regulatory function of PARP within cell growth and potentially development. PARP inhibition enhances growth of Arabidopsis thaliana by enhancing the cell number. Time course single leaf transcriptomics shows that PARP inhibition regulates a small subset of genes which are related to growth promotion, cell cycle and the control of metabolism. This is supported by metabolite analysis showing overall changes in primary and particularly secondary metabolism. Taken together the results indicate a versatile function of PARP beyond its previously reported roles in controlling plant stress tolerance and thus can be a useful target for enhancing biomass production. PMID:24587323

  20. Effect and mechanism of poly (ADP-ribose) polymerase-1 in aldosterone-induced apoptosis

    PubMed Central

    QIAO, WEIWEI; ZHANG, WEILI; SHAO, SHUHONG; GAI, YUSHENG; ZHANG, MINGXIANG

    2015-01-01

    The present study aimed to investigate the effects of aldosterone on vascular endothelial cells and the viability of poly (ADP-ribose) polymerase 1 (PARP1) in cells, and to examine the molecular mechanisms underlying the effects of aldosterone on vascular endothelial cell injury. Cultured endothelial cells were treated either with different concentrations of aldosterone for the same duration or with the same concentrations of aldosterone for different durations, and the levels of apoptosis and activity of PARP1 in the cells were detected, respectively. Aldosterone receptor antagonists or PARP1 inhibitors were added to cells during treatment with aldosterone and the levels of apoptosis and activity of PARP1 were detected. As the concentration of aldosterone increased or the treatment time increased, the number of apoptotic cells and the activity of PARP1 increased. The aldosterone receptor antagonists and PARP1 inhibitors inhibited the increase of apoptosis and PARP1 activity caused by aldosterone treatment. Aldosterone activated the activity of PARP1 via the aldosterone receptor, inhibiting cell proliferation and inducing apoptosis. Treatment with PARP1 may be used as a target for vascular diseases caused by aldosterone at high concentrations. PMID:25872931

  1. ATM deficiency sensitizes mantle cell lymphoma cells to poly(ADP-ribose) polymerase-1 inhibitors.

    PubMed

    Williamson, Chris T; Muzik, Huong; Turhan, Ali G; Zamò, Alberto; O'Connor, Mark J; Bebb, D Gwyn; Lees-Miller, Susan P

    2010-02-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) inhibition is toxic to cells with mutations in the breast and ovarian cancer susceptibility genes BRCA1 or BRCA2, a concept termed synthetic lethality. However, whether this approach is applicable to other human cancers with defects in other DNA repair genes has yet to be determined. The ataxia telangiectasia mutated (ATM) gene is altered in several human cancers including mantle cell lymphoma (MCL). Here, we characterize a panel of MCL cell lines for ATM status and function and investigate the potential for synthetic lethality in MCL in the presence of small-molecule inhibitors of PARP-1. We show that Granta-519 and UPN2 cells have low levels of ATM protein, are defective in DNA damage-induced ATM-dependent signaling, are radiation sensitive, and have cell cycle checkpoint defects: all characteristics of defective ATM function. Significantly, Granta-519 and UPN2 cells were more sensitive to PARP-1 inhibition than were the ATM-proficient MCL cell lines examined. Furthermore, the PARP-1 inhibitor olaparib (known previously as AZD2281/KU-0059436) significantly decreased tumor growth and increased overall survival in mice bearing s.c. xenografts of ATM-deficient Granta-519 cells while producing only a modest effect on overall survival of mice bearing xenografts of the ATM-proficient cell line, Z138. Thus, PARP inhibitors have therapeutic potential in the treatment of MCL, and the concept of synthetic lethality extends to human cancers with ATM alterations. PMID:20124459

  2. Involvement of poly(ADP-ribose) polymerase in paraptotic cell death of D. discoideum.

    PubMed

    Rajawat, Jyotika; Mir, Hina; Alex, Tina; Bakshi, Sonal; Begum, Rasheedunnisa

    2014-01-01

    Paraptosis is mediated by several proteins, poly(ADP-ribose) polymerase being one of them. D. discoideum lacks caspases thus providing a better system to dissect out the role of PARP in paraptosis. The cell death phenotype in unicellular eukaryote, D. discoideum is similar to the programmed cell death phenotype of multicellular animals. However, the events downstream to the death signal of PCD in D. discoideum are yet to be understood. Our results emphasize that oxidative stress in D. discoideum lacking caspases leads to PARP activation, mitochondrial membrane potential changes, followed by the release of apoptosis inducing factor from mitochondria. AIF causes large scale DNA fragmentation, a hallmark feature of paraptosis. The role of PARP in paraptosis is reiterated via PARP inhibition by benzamide, PARG inhibition by gallotannin and PARP down-regulation, which delays paraptosis. PARP, PARG and AIF interplay is quintessential in paraptosis of D. discoideum. This is the first report to establish the involvement of PARP in the absence of caspase activity in D. discoideum which could be of evolutionary significance and gives a lead to understand the caspase independent paraptotic mechanism in higher organisms. PMID:24129923

  3. Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors.

    PubMed

    Ohno, Kazuki; Mitsui, Takashi; Tanida, Yoshiaki; Matsuura, Azuma; Fujitani, Hideaki; Niimi, Tatsuya; Orita, Masaya

    2011-02-01

    Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein/ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1)/inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann/surface area (MM/PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects. PMID:20480380

  4. DNA vector-based RNAi approach for stable depletion of poly(ADP-ribose) polymerase-1

    SciTech Connect

    Shah, Rashmi G.; Ghodgaonkar, Medini M.; Affar, El Bachir; Shah, Girish M. . E-mail: girish.shah@crchul.ulaval.ca

    2005-05-27

    RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO{sub 4} protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2.

  5. Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

    PubMed

    Kivlin, Christine M; Watson, Kelsey L; Al Sannaa, Ghadah A; Belousov, Roman; Ingram, Davis R; Huang, Kai-Lieh; May, Caitlin D; Bolshakov, Svetlana; Landers, Sharon M; Kalam, Azad Abul; Slopis, John M; McCutcheon, Ian E; Pollock, Raphael E; Lev, Dina; Lazar, Alexander J; Torres, Keila E

    2016-01-01

    Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy. PMID:26650448

  6. Poly (ADP-Ribose) Polymerase Mediates Diabetes-Induced Retinal Neuropathy

    PubMed Central

    Mohammad, Ghulam; Siddiquei, Mohammad Mairaj

    2013-01-01

    Retinal neuropathy is an early event in the development of diabetic retinopathy. One of the potential enzymes that are activated by oxidative stress in the diabetic retina is poly (ADP-ribose) polymerase (PARP). We investigated the effect of the PARP inhibitor 1,5-isoquinolinediol on the expression of the neurodegeneration mediators and markers in the retinas of diabetic rats. After two weeks of streptozotocin-induced diabetes, rats were treated with 1,5-isoquinolinediol (3 mg/kg/day). After 4 weeks of diabetes, the retinas were harvested and the levels of reactive oxygen species (ROS) were determined fluorometrically and the expressions of PARP, phosporylated-ERK1/2, BDNF, synaptophysin, glutamine synthetase (GS), and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, PARP-1/2, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expressions of BDNF synaptophysin and GS were significantly decreased in the retinas of diabetic rats, compared to nondiabetic rats. Administration of 1,5-isoquinolinediol did not affect the metabolic status of the diabetic rats, but it significantly attenuated diabetes-induced upregulation of PARP, ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF, synaptophysin, and GS. These findings suggest a beneficial effect of the PARP inhibitor in increasing neurotrophic support and ameliorating early retinal neuropathy induced by diabetes. PMID:24347828

  7. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  8. A novel crosstalk between BRCA1 and poly (ADP-ribose) polymerase 1 in breast cancer

    PubMed Central

    Li, Da; Bi, Fang-Fang; Chen, Na-Na; Cao, Ji-Min; Sun, Wu-Ping; Zhou, Yi-Ming; Li, Chun-Yan; Yang, Qing

    2014-01-01

    BRCA mutations are the main known hereditary factor for breast cancer. Notably, poly (ADP-ribose) polymerase 1 (PARP1) expression status plays a critical role in breast cancer progression and the clinical development of PARP1 inhibitors to treat BRCA-mutated breast cancer has advanced rapidly. However, dynamic crosstalk between BRCA1 and PARP1 remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation, promoter methylation, or knockdown) were accompanied by increased PARP1 and nicotinamide adenine dinucleotide (NAD) levels, and a subsequent increase in NAD-dependent PARP1 activity in MDA-MB-231 and primary breast cancer cells; (ii) the overexpression of BRCA1 resulted in decreased PARP1 and NAD levels, and a subsequent impairment in NAD-dependent PARP1 activity in MDA-MB-231 and primary breast cancer cells; and (iii) intracellular NAD levels were largely responsible for regulating PARP1 activity in breast cancer cells, and NAD levels were positively correlated with PARP1 activity in human breast cancer specimens (R = 0.647, P < 0.001). Interestingly, the high efficiency of PARP1 triggered by BRCA1 inactivation may further inhibit BRCA1 transcription by NAD depletion. These results highlight a novel interaction between BRCA1 and PARP1, which may be beneficial for the dynamic balance between BRCA1 and PARP1-related biologic processes, especially for maintaining stable DNA repair ability. All of this may improve our understanding of the basic molecular mechanism underlying BRCA1- and PARP1-related breast cancer progression. PMID:25485588

  9. Neuroprotective Effects of Poly(ADP-ribose)polymerase Inhibitor Olaparib in Transient Cerebral Ischemia.

    PubMed

    Teng, Fei; Zhu, Ling; Su, Junhui; Zhang, Xi; Li, Ning; Nie, Zhiyu; Jin, Lingjing

    2016-07-01

    Olaparib was the first poly(ADP-ribose)polymerase inhibitor approved by Food and Drug Administration for oncology treatment. However, its neuroprotective effects have not been elucidated. This study aimed to evaluate the effects of olaparib in transient cerebral ischemia. A mouse model of transient middle cerebral artery occlusion was used. Reperfusion was performed at 2 h after ischemia. Different doses of olaparib (1, 3, 5, 10 and 25 mg/kg) were administered intraperitoneally immediately after reperfusion. Twenty-four hours after ischemia, the neurological score was assessed, and grip and string tests were performed to evaluate the behavioral deficits in the mice. Cresyl violet staining was used to assess cerebral edema and the lesion volume. Immunohistochemistry was performed to evaluate the expression of blood-brain barrier proteins collagen IV and claudin-5, as well as extravasation of IgG. Ischemia induced a neurological deficit, which was significantly ameliorated by olaparib at 3 and 5 mg/kg. However, this neuroprotective effect was not observed in mice treated with either low-dose or high-dose olaparib. Both 3 and 5 mg/kg olaparib markedly reduced cerebral infarction volume, but not cerebral edema. The expression of collagen IV decreased after cerebral ischemia, which was improved by olaparib at 3 and 5 mg/kg. These results were confirmed by the reduction of IgG extravasation with olaparib. Olaparib showed clear neuroprotective effects in transient ischemic mice mainly through the reduction of cerebral infarction and blood-brain barrier damage. PMID:26869042

  10. Poly(ADP-ribose) polymerase 1 modulates the lethality of CHK1 inhibitors in carcinoma cells.

    PubMed

    Mitchell, Clint; Park, Margaret; Eulitt, Patrick; Yang, Chen; Yacoub, Adly; Dent, Paul

    2010-11-01

    Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2. CHK1 inhibitors modestly increased the levels of PARP1 ADP ribosylation and molecular or small-molecule inhibition of PARP1 blocked CHK1 inhibitor-stimulated histone H2AX phosphorylation and activation of ERK1/2. Stimulated histone H2AX phosphorylation was ataxia telangiectasia-mutated protein-dependent. Multiple CHK1 inhibitors interacted in a greater than additive fashion with multiple PARP1 inhibitors to cause transformed cell-killing in short-term viability assays and synergistically killed tumor cells in colony-formation assays. Overexpression of BCL-xL or loss of BAX/BAK function, but not the function of BID, suppressed CHK1 inhibitor + PARP1 inhibitor lethality. Inhibition of BCL-2 family protein function enhanced CHK1 inhibitor + PARP1 inhibitor lethality and restored drug-induced cell-killing in cells overexpressing BCL-xL. Thus, PARP1 plays an important role in regulating the ability of CHK1 inhibitors to activate ERK1/2 and the DNA damage response. An inability of PARP1 to modulate this response results in transformed cell death mediated through the intrinsic apoptosis pathway. PMID:20696794

  11. Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells.

    PubMed

    Burgman, P; Konings, A W

    1988-12-01

    The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks. PMID:3144718

  12. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations.

    PubMed

    Natarajan, A T; van Zeeland, A A; Zwanenburg, T S

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, we have measured the influence of 3AB on DNA repair following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. Both acentric fragments and exchanges increase indicating that the presence of 3AB slows down the repair of DNA strand breaks (probably DNA double strand breaks), thus making breaks available for interaction with each other to give rise to exchanges. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. Most 3AB induced SCEs occur during the second cell cycle, in which DNA containing bromouridine (BU) is used as template for replication. BU containing DNA appears to be prone to errors during replication. The extent of increase in the frequencies of SCEs by 3AB is correlated with the amount of BU incorporated in the DNA of the cells. The frequencies of spontaneously occurring DNA single strand breaks in cells grown in BrdUrd containing medium are higher than in the cells grown in normal medium and this increase depends on the amount of BU incorporated in the DNA of these cells. We have studied the extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the

  13. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. PMID:26428915

  14. Prevention of tumorigenesis of oncogene-transformed rat fibroblasts with DNA site inhibitors of poly(ADP ribose) polymerase

    SciTech Connect

    Tseng, A. Jr.; Lee, W.M.F.; Kirsten, E.; Hakam, A.; McLick, J.; Buki, K.; Kun, E.

    1987-02-01

    The EJ-ras gene was placed under the transcriptional control of the steroid-inducible mouse mammary tumor virus promoter/enhancer and introduced into Rat-1 fibroblasts, yielding the 14C cell line. When these cells were exposed to dexamethasone in vitro, EJ-ras mRNA was induced 15- to 20-fold, the cells grew in agar, and, after injection of cells into syngenic Fischer 344 rats, they produced lethal fibrosarcomas. Inhibitors of poly(ADP ribose) polymerase, which prevent the activation of the purified enzyme by a synthrtic octadeoxyribonucleotide duplex, inhibited both in vivo tumorigenicity and in vitro growth in soft agar. The enzyme inhibitor 1,2-benzopyrone, which was studied in detail, and other polymerase inhibitors had no effect on EJ-ras mRNA or p21 protein expression. Poly(ADP ribose) polymerase was inhibited by the drug in both untreated and dexamethasone-treated cells both in vitro and in vivo to the same extent, but biological consequences of enzyme inhibition were manifest only when the cells were in the transformed tumorigenic state.

  15. Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus.

    PubMed Central

    Faraone-Mennella, M R; Gambacorta, A; Nicolaus, B; Farina, B

    1998-01-01

    A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54-55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 degreesC, and thermostable, with a half-life of 204 min at 80 degreesC, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154+/-50 microM; in the pH range 6.5-10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step. PMID:9761745

  16. Poly ADP-Ribose Polymerase Inhibition Ameliorates Hind Limb Ischemia Reperfusion Injury in a Murine Model of Type 2 Diabetes

    PubMed Central

    Long, Chandler A.; Boloum, Valy; Albadawi, Hassan; Tsai, Shirling; Yoo, Hyung-Jin; Oklu, Rahmi; Goldman, Mitchell H.; Watkins, Michael T.

    2013-01-01

    Introduction Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes; ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods db/db mice underwent 1.5hrs of hind limb ischemia followed by 1, 7, or 24hrs reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24hrs period; the untreated group received Lactated ringer’s (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity /intracellular localization and poly-ADP-ribosylation of GAPDH. Results PARP activity was significantly lower in the PJ34 treated groups compared to the LR group at 7 and 24 hours reperfusion. There was significantly less muscle fiber injury in the PJ34 treated group compared to LR treated mice at 24 hrs reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7hrs and 24hrs IR. There were significant increases in metabolic activity only at 24 hours IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly ADP-ribosylation and nuclear translocation of GAPDH. Conclusions PJ34 reduced PARP activity, GAPDH ribosylation, GAPDH translocation, ameliorated muscle fiber injury, and increased metabolic activity following hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy following IR in diabetic humans. PMID:23549425

  17. Poly(ADP-ribose) polymerase inhibitors regulate the mechanism of sulfur mustard-initiated cell death in human lymphocytes.

    PubMed

    Meier, H L; Millard, C; Moser, J

    2000-12-01

    Sulfur mustard (HD) produces slow-healing skin lesions that contain large, tight fluid-filled blisters. These lesions are the result of severe damage to areas of the body exposed to HD and require extensive medical care before complete recovery is achieved. Converting the mechanism of HD-initiated cell death from an inflammatory oncosis (homicide) to benign apoptosis (assisted suicide) may reduce the extent of cellular damage and the time required for healing. HD-exposed human lymphocytes lose cellular function, membrane integrity and viability, and suffer degradation of their nuclear components. The treatment of HD-exposed cells with poly(ADP-ribose) polymerase inhibitors prevents or alters the HD-initiated loss of cell viability, membrane integrity, cellular metabolic constituent (NAD) and cellular energy (ATP), while initiating alterations in nuclear constituents. It is hoped that by preventing or altering these HD-initiated changes we can limit the extent of the injury, decrease the time required for repair and reduce the loss of performance suffered by exposed individuals. The use of poly(ADP-ribose) polymerase inhibitors to assist in initiating apoptosis in affected cells should help to achieve these objectives while preventing the chance of further disease development later in the exposed individuals. PMID:11428651

  18. ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

    SciTech Connect

    M Langelier; J Planck; S Roy; J Pascal

    2011-12-31

    Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

  19. Mass spectrometry-based functional proteomics of poly(ADP-ribose) polymerase-1.

    PubMed

    Pic, Emilie; Gagné, Jean-Philippe; Poirier, Guy G

    2011-12-01

    PARP-1 is an abundant nuclear protein that plays an essential role in the regulation of many genome integrity and chromatin-based processes, such as DNA repair, replication or transcriptional regulation. PARP-1 modulates the function of chromatin and nuclear proteins through several poly(ADP-ribose) (pADPr)-dependent pathways. Aside from the clearly established role of PARP-1 in the maintenance of genome stability, PARP-1 also emerged as an important regulator that links chromatin functions with extranuclear compartments. pADPr signaling has notably been found to be responsible for PARP-1-mediated mitochondrial dysfunction and cell death. Defining the mechanisms that govern the intrinsic functions of PARP-1 is fundamental to the understanding of signaling networks regulated by pADPr. The emergence of mass spectrometry-based proteomics and its broad applications in the study of biological systems represents an outstanding opportunity to widen our knowledge of the functional spectrum of PARP-1. In this article, we summarize various PARP-1 targeted proteomics studies and proteome-wide analyses that shed light on its protein interaction partners, expression levels and post-translational modifications. PMID:22087659

  20. PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

    PubMed Central

    Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

    2010-01-01

    PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PMID:20228053

  1. Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2.

    PubMed

    Martin, Kayla A; Cesaroni, Matteo; Denny, Michael F; Lupey, Lena N; Tempera, Italo

    2015-12-01

    Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2. PMID:26370511

  2. Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2

    PubMed Central

    Martin, Kayla A.; Cesaroni, Matteo; Denny, Michael F.; Lupey, Lena N.

    2015-01-01

    Posttranslational modifications, such as poly(ADP-ribosyl)ation (PARylation), regulate chromatin-modifying enzymes, ultimately affecting gene expression. This study explores the role of poly(ADP-ribose) polymerase (PARP) on global gene expression in a lymphoblastoid B cell line. We found that inhibition of PARP catalytic activity with olaparib resulted in global gene deregulation, affecting approximately 11% of the genes expressed. Gene ontology analysis revealed that PARP could exert these effects through transcription factors and chromatin-remodeling enzymes, including the polycomb repressive complex 2 (PRC2) member EZH2. EZH2 mediates the trimethylation of histone H3 at lysine 27 (H3K27me3), a modification associated with chromatin compaction and gene silencing. Both pharmacological inhibition of PARP and knockdown of PARP1 induced the expression of EZH2, which resulted in increased global H3K27me3. Chromatin immunoprecipitation confirmed that PARP1 inhibition led to H3K27me3 deposition at EZH2 target genes, which resulted in gene silencing. Moreover, increased EZH2 expression is attributed to the loss of the occupancy of the transcription repressor E2F4 at the EZH2 promoter following PARP inhibition. Together, these data show that PARP plays an important role in global gene regulation and identifies for the first time a direct role of PARP1 in regulating the expression and function of EZH2. PMID:26370511

  3. Coordinated Regulation of TIP60 and Poly(ADP-Ribose) Polymerase 1 in Damaged-Chromatin Dynamics.

    PubMed

    Ikura, Masae; Furuya, Kanji; Fukuto, Atsuhiko; Matsuda, Ryo; Adachi, Jun; Matsuda, Tomonari; Kakizuka, Akira; Ikura, Tsuyoshi

    2016-05-15

    The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses. PMID:26976643

  4. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. )

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  5. p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

    PubMed Central

    Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A.; Scovassi, A. Ivana; Lavrik, Olga; Prosperi, Ennio

    2016-01-01

    The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis. PMID:26730949

  6. Caffeine metabolites are inhibitors of the nuclear enzyme poly(ADP-ribose)polymerase-1 at physiological concentrations.

    PubMed

    Geraets, Liesbeth; Moonen, Harald J J; Wouters, Emiel F M; Bast, Aalt; Hageman, Geja J

    2006-09-28

    The activity of the nuclear enzyme poly(ADP-ribose)polymerase-1 (E.C.2.4.2.30), which is highly activated by DNA strand breaks, is associated with the pathophysiology of both acute as well as chronic inflammatory diseases. PARP-1 overactivation and the subsequent extensive turnover of its substrate NAD+ put a large demand on mitochondrial ATP-production. Furthermore, due to its reported role in NF-kappaB and AP-1 mediated production of pro-inflammatory cytokines, PARP-1 is considered an interesting target in the treatment of these diseases. In this study the PARP-1 inhibiting capacity of caffeine and several metabolites as well as other (methyl)xanthines was tested using an ELISA-assay with purified human PARP-1. Caffeine itself showed only weak PARP-1 inhibiting activity, whereas the caffeine metabolites 1,7-dimethylxanthine, 3-methylxanthine and 1-methylxanthine, as well as theobromine and theophylline showed significant PARP-1 inhibiting activity. Further evaluation of these compounds in H2O2-treated A549 lung epithelial and RF24 vascular endothelial cells revealed that the decrease in NAD+-levels as well as the formation of the poly(ADP-ribose)polymer was significantly prevented by the major caffeine metabolite 1,7-dimethylxanthine. Furthermore, H2O2-induced necrosis could be prevented by a high dose of 1,7-dimethylxanthine. Finally, antioxidant effects of the methylxanthines could be ruled out with ESR and measurement of the TEAC. Concluding, caffeine metabolites are inhibitors of PARP-1 and the major caffeine metabolite 1,7-dimethylxanthine has significant PARP-1 inhibiting activity in cultured epithelial and endothelial cells at physiological concentrations. This inhibition could have important implications for nutritional treatment of acute and chronic inflammatory pathologies, like prevention of ischemia-reperfusion injury or vascular complications in diabetes. PMID:16870158

  7. Pathophysiological role of poly(ADP-ribose) polymerase (PARP) activation during acetaminophen-induced liver cell necrosis in mice.

    PubMed

    Cover, Cathleen; Fickert, Peter; Knight, Tamara R; Fuchsbichler, Andrea; Farhood, Anwar; Trauner, Michael; Jaeschke, Hartmut

    2005-03-01

    DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP-/-). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP-/- mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP-/- mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition. PMID:15601672

  8. Prolonged poly(ADP-ribose) polymerase-1 activity regulates JP-8-induced sustained cytokine expression in alveolar macrophages.

    PubMed

    Espinoza, Luis A; Smulson, Mark E; Chen, Zun

    2007-05-01

    Environmental pollutants inducing oxidative stress stimulate chronic inflammatory responses in the lung leading to pulmonary tissue dysfunction. In response to oxidative stress, alveolar macrophages produce both reactive oxygen species and reactive nitrogen species, which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of alveolar macrophages to a nonlethal dose (8 microg/ml) of JP-8, the kerosene-based hydrocarbon jet fuel, induced the persistent expression of IL-1, iNOS, and COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). Because poly(ADP-ribose) polymerase (PARP-1), a coactivator of NF-kappaB, regulates inflammatory responses and associated disorders in the airways, we determined whether JP-8 induces the poly(ADP-ribosyl)ation automodification of PARP-1 in alveolar macrophages. We observed that PARP-1 is activated in a time-dependent manner, which was temporally coincident with the prolonged activation of NF-kappaB and with the augmented expression of the proinflammatory factors described above. The 4 microg/ml dilution of JP-8 also increased the activity of PARP-1 as well as the expression of iNOS and COX-2, indicating that lower doses of JP-8 also affect the regulation of proinflammatory factors in pulmonary macrophages. Together, these results demonstrate that an extensive induction of PARP-1 might coordinate the persistent expression of proinflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury from occupational exposure. PMID:17395016

  9. Regulation of Epstein-Barr virus OriP replication by poly(ADP-ribose) polymerase 1.

    PubMed

    Tempera, Italo; Deng, Zhong; Atanasiu, Constandache; Chen, Chi-Ju; D'Erme, Maria; Lieberman, Paul M

    2010-05-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant, chromatin-associated, NAD-dependent enzyme that functions in multiple chromosomal processes, including DNA replication and chromatin remodeling. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is a dynamic genetic element that confers stable episome maintenance, DNA replication initiation, and chromatin organization functions. OriP function depends on the EBV-encoded origin binding protein EBNA1. We have previously shown that EBNA1 is subject to negative regulation by poly(ADP-ribosyl)ation (PARylation). We now show that PARP1 physically associates with OriP in latently EBV-infected B cells. Short hairpin RNA depletion of PARP1 enhances OriP replication activity and increases EBNA1, origin recognition complex 2 (ORC2), and minichromosome maintenance complex (MCM) association with OriP. Pharmacological inhibitors of PARP1 enhance OriP plasmid maintenance and increase EBNA1, ORC2, and MCM3 occupancy at OriP. PARylation in vitro inhibits ORC2 recruitment and remodels telomere repeat factor (TRF) binding at the dyad symmetry (DS) element of OriP. Purified PARP1 can ribosylate EBNA1 at multiple sites throughout its amino terminus but not in the carboxy-terminal DNA binding domain. We also show that EBNA1 linking regions (LR1 and LR2) can bind directly to oligomers of PAR. We propose that PARP1-dependent PARylation of EBNA1 and adjacently bound TRF2 induces structural changes at the DS element that reduce EBNA1 DNA binding affinity and functional recruitment of ORC. PMID:20219917

  10. Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

    PubMed

    Shrestha, Elina; Hussein, Maryem A; Savas, Jeffery N; Ouimet, Mireille; Barrett, Tessa J; Leone, Sarah; Yates, John R; Moore, Kathryn J; Fisher, Edward A; Garabedian, Michael J

    2016-05-20

    Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis. PMID:27026705

  11. Inhibition of poly(ADP-ribose) polymerase-1 by arsenite interferes with repair of oxidative DNA damage.

    PubMed

    Ding, Wei; Liu, Wenlan; Cooper, Karen L; Qin, Xu-Jun; de Souza Bergo, Patrícia L; Hudson, Laurie G; Liu, Ke Jian

    2009-03-13

    Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2'-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly(ADP-ribosyl)ation is one likely mechanism for the reported co-carcinogenic activities of arsenic in UVR-induced skin carcinogenesis. PMID:19056730

  12. Poly(ADP-Ribose) Polymerase in Cervical Cancer Pathogenesis: Mechanism and Potential Role for PARP Inhibitors.

    PubMed

    Kotsopoulos, Ioannis C; Kucukmetin, Ali; Mukhopadhyay, Asima; Lunec, John; Curtin, Nicola J

    2016-05-01

    Treatment options for disease recurrence of women treated for locally advanced and advanced cervical cancer are very limited-largely palliative chemotherapy. The low efficacy of the currently available drugs raises the need for new targeted agents. Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutic agents in cancers associated with defects in DNA repair. Their therapeutic potential in cervical cancer is currently being evaluated in 3 ongoing clinical trials. Here we review the available information regarding all the aspects of PARP in cervical intraepithelial neoplasia and invasive cervical cancer, from expression and the mechanism of action to the role of the polymorphisms in the pathogenesis of the disease, as well as the potential of the inhibitors. We finally propose a new unifying theory regarding the role of PARPs in the development of cervical carcinomas. PMID:26905326

  13. Structural basis for lack of ADP-ribosyltransferase activity in poly(ADP-ribose) polymerase-13/zinc finger antiviral protein.

    PubMed

    Karlberg, Tobias; Klepsch, Mirjam; Thorsell, Ann-Gerd; Andersson, C David; Linusson, Anna; Schüler, Herwig

    2015-03-20

    The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity. PMID:25635049

  14. Poly(ADP-ribose) polymerase activation induces high mobility group box 1 release from proximal tubular cells during cisplatin nephrotoxicity.

    PubMed

    Kim, J

    2016-06-20

    Cisplatin is one of the most potent chemotherapy drugs against cancer, but its major side effect such as nephrotoxicity limits its use. Inhibition of poly(ADP-ribose) polymerase (PARP) protects against various renal diseases via gene transactivation and/or ADP-ribosylation. However, the role of PARP in necrotic cell death during cisplatin nephrotoxicity remains an open question. Here we demonstrated that pharmacological inhibition of PARP by postconditioning dose-dependently prevented tubular injury and renal dysfunction following cisplatin administration in mice. PARP inhibition by postconditioning also attenuated ATP depletion during cisplatin nephrotoxicity. Systemic release of high mobility group box 1 (HMGB1) protein in plasma induced by cisplatin administration was significantly diminished by PARP inhibition by postconditioning. In in vitro kidney proximal tubular cell lines, PARP inhibition by postconditioning also diminished HMGB1 release from cells. These data demonstrate that cisplatin-induced PARP1 activation contributes to HMGB1 release from kidney proximal tubular cells, resulting in the promotion of inflammation during cisplatin nephrotoxicity. PMID:26447520

  15. 3-aminobenzamide, one of poly(ADP-ribose)polymerase-1 inhibitors, rescuesapoptosisin rat models of spinal cord injury

    PubMed Central

    Meng, Xianqing; Song, Wenqi; Deng, Bin; Xing, Ziling; Zhang, Weihong

    2015-01-01

    Poly(ADP-ribose)polymerase-1 (PARP-1) is anubiquitous, DNA repair-associated enzyme, which participates in gene expression, cell death, central nerve system (CNS) disorders and oxidative stress. According to the previous studies, PARP-1 over-activation may lead to over-consumption of ATP and even cell apoptosis. Spinal cord injury (SCI) is an inducement towards PARP-1 over-activation due to its massive damage to DNA. 3-aminobenzamide (3-AB) is a kind of PARP-1 inhibitors. The relationship among PARP-1, 3-AB, SCI and apoptosis has not been fully understood. Hence, in the present study, we focused on the effects of 3-AB on cell apoptosis after SCI. Accordingly, SCI model was constructed artificially, and 3-AB was injected intrathecally into the Sprague-Dawley (SD) rats. The results demonstrated an increase in cell apoptosis after SCI. Furthermore, PARP-1 was over-activated after SCI but inhibited by 3-AB injection. In addition, apoptosis-inducing factor (AIF) was inhibited but B-cell lymphoma-2 (Bcl-2) was up-regulated by 3-AB. Interestingly, caspase-3 was not significantly altered with or without 3-AB. In conclusion, our experiments showed that 3-AB, as a PARP-1 inhibitor, could inhibit cell apoptosis after SCI in caspase-independent way, which could provide a better therapeutic target for the treatment of SCI. PMID:26722405

  16. Continuous inhibition of poly(ADP-ribose) polymerase does not reduce reperfusion injury in isolated rat heart.

    PubMed

    Nishizawa, Kenya; Yanagida, Shigeki; Yamagishi, Tadashi; Takayama, Eiichi; Bessho, Motoaki; Kusuhara, Masatoshi; Adachi, Takeshi; Ohsuzu, Fumitaka

    2013-07-01

    Poly(ADP-ribose) polymerase (PARP), an enzyme that is important to the regulation of nuclear function, is activated by DNA strand breakage. In massive DNA damage, PARP is overactivated, exhausting nicotinamide adenine dinucleotide and leading to cell death. Recent studies have succeeded in reducing cellular damage in ischemia/reperfusion by inhibiting PARP. However, PARP plays an important part in the DNA repair system, and its inhibition may be hazardous in certain situations. We compared the short-time inhibition of PARP against continuous inhibition during ischemia/reperfusion using isolated rat hearts. The hearts were reperfused after 21 minutes of ischemia with a bolus injection of 3-aminobenzamide (3-AB) (10 mg/kg) followed by continuous 3-AB infusion (50 μM) for the whole reperfusion period or for the first 6 minutes or without 3-AB. At the end of reperfusion, contractile function, high-energy phosphate content, nicotinamide adenine dinucleotide content, and infarcted area were significantly preserved in the 3-AB 6-minute group. In the 3-AB continuous group, these advantages were not apparent. At the end of reperfusion, PARP cleavage had significantly proceeded in the 3-AB continuous group, indicating initiation of the apoptotic cascade. Thus, continuous PARP inhibition by 3-AB does not reduce reperfusion injury in the isolated rat heart, which may be because of acceleration of apoptosis. PMID:23846805

  17. Natural and glucosyl flavonoids inhibit poly(ADP-ribose) polymerase activity and induce synthetic lethality in BRCA mutant cells

    PubMed Central

    MAEDA, JUNKO; ROYBAL, ERICA J.; BRENTS, COLLEEN A.; UESAKA, MITSURU; AIZAWA, YASUSHI; KATO, TAKAMITSU A.

    2014-01-01

    Poly(ADP-ribose) polymerase (PARP) inhibitors have been proven to represent superior clinical agents targeting DNA repair mechanisms in cancer therapy. We investigated PARP inhibitory effects of the natural and synthetic flavonoids (quercetin, rutin, monoglucosyl rutin and maltooligosyl rutin) and tested the synthetic lethality in BRCA2 mutated cells. In vitro ELISA assay suggested that the flavonoids have inhibitory effects on PARP activity, but glucosyl modifications reduced the inhibitory effect. Cytotoxicity tests of Chinese hamster cells defective in BRCA2 gene (V-C8) and its parental V79 cells showed BRCA2-dependent synthetic lethality when treated with the flavonoids. BRCA2 mutated cells were three times more sensitive to the flavonoids than the wild-type and gene complemented cells. Reduced toxicity was observed in a glucosyl modification-dependent manner. The present study provides support for the clinical use of new treatment drugs, and is the beginning of the potential application of flavonoids in cancer prevention and the periodic consumption of appropriate flavonoids to reduce cancer risk in individuals carrying a mutant allele of the BRCA2 gene. PMID:24317580

  18. Effectiveness and safety of poly (ADP-ribose) polymerase inhibitors in cancer therapy: A systematic review and meta-analysis.

    PubMed

    Bao, Zhengqiang; Cao, Chao; Geng, Xinwei; Tian, Baoping; Wu, Yanping; Zhang, Chao; Chen, Zhihua; Li, Wen; Shen, Huahao; Ying, Songmin

    2016-02-16

    Poly (ADP-ribose) polymerase (PARP) inhibitors are a class of small-molecule drugs suppressing PARP enzymes activity, inducing the death of cells deficient in homologous recombination repair (HRR). HRR deficiency is common in tumor cells with BRCA gene mutation. Since their first clinical trial in 2003, PARP inhibitors have shown benefit in the treatment of HRR-deficient tumors. Recently, several randomized clinical trials (RCTs) have been conducted to investigate the potential benefit of administration of PARP inhibitors in cancer patients. However, the results remain controversial. To evaluate the efficiency and safety of PARP inhibitors in patients with cancer, we performed a comprehensive meta-analysis of RCTs. According to our study, PARP inhibitors could clearly improve progression-free survival (PFS), especially in patients with BRCA mutation. However, our study showed no significant difference in overall survival (OS) between the PARP inhibitors and controls, even in the BRCA mutation group. Little toxicity was reported in the rate of treatment correlated adverse events (AEs) in PARP inhibitor group compared with controls. In conclusion, PARP inhibitors do well in improving PFS with little toxicity, especially in patients with BRCA deficiency. PMID:26399274

  19. Effectiveness and safety of poly (ADP-ribose) polymerase inhibitors in cancer therapy: A systematic review and meta-analysis

    PubMed Central

    Geng, Xinwei; Tian, Baoping; Wu, Yanping; Zhang, Chao; Chen, Zhihua; Li, Wen; Shen, Huahao; Ying, Songmin

    2016-01-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors are a class of small-molecule drugs suppressing PARP enzymes activity, inducing the death of cells deficient in homologous recombination repair (HRR). HRR deficiency is common in tumor cells with BRCA gene mutation. Since their first clinical trial in 2003, PARP inhibitors have shown benefit in the treatment of HRR-deficient tumors. Recently, several randomized clinical trials (RCTs) have been conducted to investigate the potential benefit of administration of PARP inhibitors in cancer patients. However, the results remain controversial. To evaluate the efficiency and safety of PARP inhibitors in patients with cancer, we performed a comprehensive meta-analysis of RCTs. According to our study, PARP inhibitors could clearly improve progression-free survival (PFS), especially in patients with BRCA mutation. However, our study showed no significant difference in overall survival (OS) between the PARP inhibitors and controls, even in the BRCA mutation group. Little toxicity was reported in the rate of treatment correlated adverse events (AEs) in PARP inhibitor group compared with controls. In conclusion, PARP inhibitors do well in improving PFS with little toxicity, especially in patients with BRCA deficiency. PMID:26399274

  20. Synthesis and Evaluation of a Radioiodinated Tracer with Specificity for Poly(ADP-ribose) Polymerase-1 (PARP-1) in Vivo.

    PubMed

    Zmuda, Filip; Malviya, Gaurav; Blair, Adele; Boyd, Marie; Chalmers, Anthony J; Sutherland, Andrew; Pimlott, Sally L

    2015-11-12

    Interest in nuclear imaging of poly(ADP-ribose) polymerase-1 (PARP-1) has grown in recent years due to the ability of PARP-1 to act as a biomarker for glioblastoma and increased clinical use of PARP-1 inhibitors. This study reports the identification of a lead iodinated analog 5 of the clinical PARP-1 inhibitor olaparib as a potential single-photon emission computed tomography (SPECT) imaging agent. Compound 5 was shown to be a potent PARP-1 inhibitor in cell-free and cellular assays, and it exhibited mouse plasma stability but approximately 3-fold greater intrinsic clearance when compared to olaparib. An (123)I-labeled version of 5 was generated using solid state halogen exchange methodology. Ex vivo biodistribution studies of [(123)I]5 in mice bearing subcutaneous glioblastoma xenografts revealed that the tracer had the ability to be retained in tumor tissue and bind to PARP-1 with specificity. These findings support further investigations of [(123)I]5 as a noninvasive PARP-1 SPECT imaging agent. PMID:26469301

  1. Regulation of poly(ADP-ribose) polymerase 1 activity by the phosphorylation state of the nuclear NAD biosynthetic enzyme NMN adenylyl transferase 1

    PubMed Central

    Berger, Felicitas; Lau, Corinna; Ziegler, Mathias

    2007-01-01

    Nuclear NAD+ metabolism constitutes a major component of signaling pathways. It includes NAD+-dependent protein deacetylation by members of the Sir2 family and protein modification by poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 has emerged as an important mediator of processes involving DNA rearrangements. High-affinity binding to breaks in DNA activates PARP-1, which attaches poly(ADP-ribose) (PAR) to target proteins. NMN adenylyl transferases (NMNATs) catalyze the final step of NAD+ biosynthesis. We report here that the nuclear isoform NMNAT-1 stimulates PARP-1 activity and binds to PAR. Its overexpression in HeLa cells promotes the relocation of apoptosis-inducing factor from the mitochondria to the nucleus, a process known to depend on poly(ADP-ribosyl)ation. Moreover, NMNAT-1 is subject to phosphorylation by protein kinase C, resulting in reduced binding to PAR. Mimicking phosphorylation, substitution of the target serine residue by aspartate precludes PAR binding and stimulation of PARP-1. We conclude that, depending on its state of phosphorylation, NMNAT-1 binds to activated, automodifying PARP-1 and thereby amplifies poly(ADP-ribosyl)ation. PMID:17360427

  2. Poly(ADP-ribose) polymerase inhibition reverses vascular dysfunction after {gamma}-irradiation

    SciTech Connect

    Beller, Carsten J. . E-mail: Carsten.Beller@urz.uni-heidelberg.de; Radovits, Tamas; Seres, Leila; Kosse, Jens; Krempien, Robert; Gross, Marie-Luise; Penzel, Roland; Berger, Irina; Huber, Peter E.; Hagl, Siegfried; Szabo, Csaba; Szabo, Gabor

    2006-08-01

    Purpose: The generation of reactive oxygen species during {gamma}-irradiation may induce DNA damage, leading to activation of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) culminating in endothelial dysfunction. In the present study, we assessed the effect of PARP inhibition on changes in vascular function after acute and short-term irradiation. Methods and Materials: In the acute experiments, aortic rings were exposed to 20 Gy of {gamma}-irradiation. The aortae were harvested after 1 or 7 days. Two additional groups received the ultrapotent PARP inhibitor, INO-1001, for 1 or 7 days after irradiation. The aortic rings were precontracted by phenylephrine and relaxation to acetylcholine and sodium nitroprusside were studied. Results: The vasoconstrictor response to phenylephrine was significantly lower both acutely and 1 and 7 days after irradiation. Vasorelaxation to acetylcholine and sodium nitroprusside was not impaired acutely after irradiation. One and seven days after irradiation, vasorelaxation to acetylcholine and sodium nitroprusside was significantly enhanced. Treatment with INO-1001 reversed vascular dysfunction after irradiation. Conclusion: Vascular dysfunction was observed 1 and 7 days after irradiation, as evidenced by reduced vasoconstriction, coupled with endothelium-dependent and -independent hyperrelaxation. PARP inhibition restored vascular function and may, therefore, be suitable to reverse vascular dysfunction after irradiation.

  3. Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice.

    PubMed

    Larmonier, Claire B; Shehab, Kareem W; Laubitz, Daniel; Jamwal, Deepa R; Ghishan, Fayez K; Kiela, Pawel R

    2016-04-22

    Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection. PMID:26912654

  4. Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

    SciTech Connect

    Gebhard, Catherine; Staehli, Barbara E.; Shi, Yi; Camici, Giovanni G.; Akhmedov, Alexander; Hoegger, Lisa; Lohmann, Christine; Matter, Christian M.; Hassa, Paul O.; Hottiger, Michael O.; Malinski, Tadeusz; Luescher, Thomas F.; and others

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. Black-Right-Pointing-Pointer PARP-1 protects from oxidative stress induced endothelial dysfunction. Black-Right-Pointing-Pointer This effect is mediated through inhibition of vasoconstrictor prostanoid production. Black-Right-Pointing-Pointer Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.

  5. Latonduine Analogs Restore F508del-Cystic Fibrosis Transmembrane Conductance Regulator Trafficking through the Modulation of Poly-ADP Ribose Polymerase 3 and Poly-ADP Ribose Polymerase 16 Activity.

    PubMed

    Carlile, Graeme W; Robert, Renaud; Matthes, Elizabeth; Yang, Qi; Solari, Roberto; Hatley, Richard; Edge, Colin M; Hanrahan, John W; Andersen, Raymond; Thomas, David Y; Birault, Véronique

    2016-08-01

    Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action. PMID:27193581

  6. Radiosensitization of Pancreatic Cancer Cells In Vitro and In Vivo through Poly (ADP-ribose) Polymerase Inhibition with ABT-888

    PubMed Central

    Tuli, Richard; Surmak, Andrew J.; Reyes, Juvenal; Armour, Michael; Hacker-Prietz, Amy; Wong, John; DeWeese, Theodore L.; Herman, Joseph M.

    2014-01-01

    OBJECTIVES To determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs. Methods Pancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25 mg/kg), radiation (5 Gy), both, or no treatment. Mice were monitored with bioluminescence imaging. RESULTS In vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8 days (P < .01). Co-treatment with 5 Gy and 1, 10 or 100 μmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 μmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39 days, and survival at 60 days of 0%, 0% and 40%, respectively. CONCLUSIONS ABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer. PMID:24836647

  7. Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression*

    PubMed Central

    Meng, X. Wei; Koh, Brian D.; Zhang, Jin-San; Flatten, Karen S.; Schneider, Paula A.; Billadeau, Daniel D.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.

    2014-01-01

    Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation. PMID:24895135

  8. Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

    SciTech Connect

    Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

    2008-11-15

    Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence of this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.

  9. Poly(ADP-ribose) polymerase inhibition combined with irradiation: A dual treatment concept to prevent neointimal hyperplasia after endarterectomy

    SciTech Connect

    Beller, Carsten J. . E-mail: Carsten.Beller@urz.uni-heidelberg.de; Kosse, Jens; Radovits, Tamas; Geroe, Domokos; Krempien, Robert; Gross, Marie-Luise; Berger, Irina; Hagl, Siegfried; Szabo, Csaba; Szabo, Gabor

    2006-11-01

    Purpose: In a rat model of endarterectomy we investigated the potential role of the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway in neointima formation and the effects of irradiation, pharmacologic inhibition of PARP, or combined pharmacologic inhibition of PARP and irradiation on vascular remodeling. Methods and Materials: Carotid endarterectomy was performed by incision of the left carotid artery with removal of intima in Sprague-Dawley rats. Six groups were studied: sham-operated rats (n = 10), control endarterectomized rats (n = 10), or endarterectomized rats irradiated with 15 Gy (n = 10), or treated with PARP inhibitor, INO-1001 (5 mg/kg/day) (n = 10), or with combined treatment with INO-1001 and irradiation with 5 Gy (n = 10) or with 15 Gy (n = 10). After 21 days, neointima formation and vascular remodeling were assessed. Results: Neointima formation after endarterectomy was inhibited by postoperative irradiation with 15 Gy and was attenuated by PARP inhibition. However, in parallel to inhibition of neointimal hyperplasia, activation of the peroxynitrite-PARP pathway in the outer vessel wall layers was triggered by postoperative irradiation. Combined pharmacologic PARP inhibition and irradiation with 15 Gy significantly reduced both neointimal hyperplasia and activation of the peroxynitrite-PARP pathway in the outer vessel wall layers. Combination of PARP inhibition and irradiation with 5 Gy was less effective than both PARP inhibition or irradiation with 15 Gy alone. Conclusions: We conclude, that combined PARP inhibition and irradiation with 15 Gy may be a new dual strategy for prevention of restenosis after surgical vessel reconstruction: combining the strong antiproliferative effect of irradiation and ameliorating irradiation-induced side effects caused by excessive PARP activation.

  10. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  11. Rapamycin-Resistant Poly (ADP-Ribose) Polymerase-1 Overexpression Is a Potential Therapeutic Target in Lymphangioleiomyomatosis

    PubMed Central

    Sun, Yang; Gallacchi, Dana; Zhang, Erik Y.; Reynolds, Samuel B.; Robinson, Lauren; Malinowska, Izabela A.; Chiou, Terry T.; Pereira, Ana M.; Li, Chenggang; Kwiatkowski, David J.

    2014-01-01

    Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2−) cells. We analyzed publicly available expression arrays of TSC2− cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2− cells. We identified the up-regulation of PARP1 in TSC2− cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2+] cells). The transcript levels of PARP1 in TSC2− cells were not affected by rapamycin. PARP1 levels were increased in TSC2− cells, xenograft tumors of rat-derived TSC2− cells, renal cystadenomas from Tsc2+/− mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2− cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2+ cells. TSC2− cells exhibit higher levels of PARP1 relative to TSC2+ cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2− cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation. PMID:24874429

  12. Poly(ADP-ribose): An organizer of cellular architecture

    PubMed Central

    2014-01-01

    Distinct properties of poly(ADP-ribose)—including its structural diversity, nucleation potential, and low complexity, polyvalent, highly charged nature—could contribute to organizing cellular architectures. Emergent data indicate that poly(ADP-ribose) aids in the formation of nonmembranous structures, such as DNA repair foci, spindle poles, and RNA granules. Informatics analyses reported here show that RNA granule proteins enriched for low complexity regions, which aid self-assembly, are preferentially modified by poly(ADP-ribose), indicating how poly(ADP-ribose) could direct cellular organization. PMID:24914234

  13. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes.

    PubMed

    Gagné, Jean-Philippe; Isabelle, Maxim; Lo, Ken Sin; Bourassa, Sylvie; Hendzel, Michael J; Dawson, Valina L; Dawson, Ted M; Poirier, Guy G

    2008-12-01

    Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli. PMID:18981049

  14. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

    PubMed Central

    Gagné, Jean-Philippe; Isabelle, Maxim; Lo, Ken Sin; Bourassa, Sylvie; Hendzel, Michael J.; Dawson, Valina L.; Dawson, Ted M.; Poirier, Guy G.

    2008-01-01

    Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli. PMID:18981049

  15. Multiple receptor conformation docking, dock pose clustering and 3D QSAR studies on human poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.

    PubMed

    Fatima, Sabiha; Jatavath, Mohan Babu; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

    2014-10-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) functions as a DNA damage sensor and signaling molecule. It plays a vital role in the repair of DNA strand breaks induced by radiation and chemotherapeutic drugs; inhibitors of this enzyme have the potential to improve cancer chemotherapy or radiotherapy. Three-dimensional quantitative structure activity relationship (3D QSAR) models were developed using comparative molecular field analysis, comparative molecular similarity indices analysis and docking studies. A set of 88 molecules were docked into the active site of six X-ray crystal structures of poly(ADP-ribose)polymerase-1 (PARP-1), by a procedure called multiple receptor conformation docking (MRCD), in order to improve the 3D QSAR models through the analysis of binding conformations. The docked poses were clustered to obtain the best receptor binding conformation. These dock poses from clustering were used for 3D QSAR analysis. Based on MRCD and QSAR information, some key features have been identified that explain the observed variance in the activity. Two receptor-based QSAR models were generated; these models showed good internal and external statistical reliability that is evident from the [Formula: see text], [Formula: see text] and [Formula: see text]. The identified key features enabled us to design new PARP-1 inhibitors. PMID:25046176

  16. Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication.

    PubMed Central

    Dantzer, F; Nasheuer, H P; Vonesch, J L; de Murcia, G; Ménissier-de Murcia, J

    1998-01-01

    Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template. PMID:9518481

  17. Poly(ADP-ribose) (PAR) polymer is a death signal

    PubMed Central

    Andrabi, Shaida A.; Kim, No Soo; Yu, Seong-Woon; Wang, Hongmin; Koh, David W.; Sasaki, Masayuki; Klaus, Judith A.; Otsuka, Takashi; Zhang, Zhizheng; Koehler, Raymond C.; Hurn, Patricia D.; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2006-01-01

    Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury. PMID:17116882

  18. Effects of abamectin exposure on male fertility in rats: potential role of oxidative stress-mediated poly(ADP-ribose) polymerase (PARP) activation.

    PubMed

    Celik-Ozenci, Ciler; Tasatargil, Arda; Tekcan, Merih; Sati, Leyla; Gungor, Ece; Isbir, Mehmet; Demir, Ramazan

    2011-12-01

    Despite the known adverse effects of abamectin pesticide, little is known about its action on male fertility. To explore the effects of exposure to abamectin on male fertility and its mechanism, low (1mg/kg/day) and high dose (4 mg/kg/day) abamectin were applied to male rats by oral gavage for 1week and for 6weeks. Weight of testes, serum reproductive hormone levels, sperm dynamics and histopathology of testes were used to evaluate the reproductive efficiency of abamectin-exposed rats. Abamectin level was determined at high concentrations in plasma and testicular tissues of male rats exposed to this pesticide. The testes weights of animals and serum testosterone concentrations did not show any significant changes after abamectin exposure. Abamectin administration was associated with decreased sperm count and motility and increased seminiferous tubule damage. In addition, significant elevations in the 4-hydroxy-2-nonenal (4-HNE)-modified proteins and poly(ADP-ribose) (PAR) expression, as markers for oxidative stress and poly(ADP-ribose) polymerase (PARP) activation, were observed in testes of rats exposed to abamectin. These results showed that abamectin exposure induces testicular damage and affects sperm dynamics. Oxidative stress-mediated PARP activation might be one of the possible mechanism(s) underlying testicular damage induced by abamectin. PMID:21945325

  19. Poly (ADP-ribose) (PAR) Binding to Apoptosis-Inducing Factor Is Critical For PAR Polymerase-1-Dependent Cell Death (Parthanatos)

    PubMed Central

    Wang, Yingfei; Kim, No Soo; Haince, Jean-Francois; Kang, HoChul; David, Karen K.; Andrabi, Shaida A.; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1. PMID:21467298

  20. Conformational Activation of Poly(ADP-ribose) Polymerase-1 upon DNA Binding Revealed by Small-Angle X-ray Scattering

    PubMed Central

    2015-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that plays key roles in several fundamental cellular processes. PARP-1 catalyzes the polymerization of nicotinamide adenine dinucleotide on itself and other acceptor proteins, forming long branched poly(ADP-ribose) polymers. The catalytic activity of PARP-1 is stimulated upon binding to damaged DNA, but how this signal is transmitted from the N-terminal DNA binding domain to the C-terminal catalytic domain in the context of the full-length enzyme is unknown. In this paper, small-angle X-ray scattering experiments and molecular dynamics simulations were used to gain insight into the conformational changes that occur during the catalytic activation of PARP-1 by an 8-mer DNA ligand. The data are consistent with a model in which binding of the DNA ligand establishes interdomain interactions between the DNA binding and catalytic domains, which induces an allosteric change in the active site that promotes catalysis. Moreover, the PARP-1–8-mer complex is seen to adopt a conformation that is poised to recruit DNA repair factors to the site of DNA damage. This study provides the first structural information about the DNA-induced conformational activation of full-length PARP-1. PMID:24588584

  1. Stable and Reusable Electrochemical Biosensor for Poly(ADP-ribose) Polymerase and Its Inhibitor Based on Enzyme-Initiated Auto-PARylation.

    PubMed

    Xu, Yuanyuan; Liu, Li; Wang, Zhaoyin; Dai, Zhihui

    2016-07-27

    A stable and reusable electrochemical biosensor for the label-free detection of poly(ADP-ribose) polymerase (PARP) is designed in this work. C-kit-1, a thiol-modified G-quadruplex oligonucleotide, is first self-assembled on a gold electrode surface. The G-quadruplex structure of c-kit-1 can specifically tether and activate PARP, resulting in the generation of negatively charged poly(ADP-ribose) polymer (PAR). On the basis of electrostatic attraction, PAR facilitates the surface accumulation of positively charged electrochemical signal molecules. Through the characterization of electrochemical signal molecules, the label-free quantification of PARP is simply implemented. On the basis of the proposed method, selective quantification of PARP can be achieved over the linear range from 0.01 to 1 U with a calculated detection limit of 0.003U. Further studies also demonstrate the applicability of the proposed method to biosamples revealing the broad potential in practical applications. Furthermore, inhibitor of PARP has also been detected with this biosensor. Meanwhile, benefited from self-assembly on solid surface, this biosensor possesses two important features, i.e., reusability and stability, which are desirable in related biosensors. PMID:27367274

  2. GDP-Mannose-4,6-Dehydratase Is a Cytosolic Partner of Tankyrase 1 That Inhibits Its Poly(ADP-Ribose) Polymerase Activity

    PubMed Central

    Bisht, Kamlesh K.; Dudognon, Charles; Chang, William G.; Sokol, Ethan S.; Ramirez, Alejandro

    2012-01-01

    Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed. PMID:22645305

  3. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    PubMed Central

    Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

    2014-01-01

    Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity. PMID:25195882

  4. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone.

    PubMed

    Aoyagi-Scharber, Mika; Gardberg, Anna S; Yip, Bryan K; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A

    2014-09-01

    Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity. PMID:25195882

  5. Targeting poly(ADP-ribose)polymerase1 in neurological diseases: A promising trove for new pharmacological interventions to enter clinical translation.

    PubMed

    Sriram, Chandra Shekhar; Jangra, Ashok; Kasala, Eshvendar Reddy; Bodduluru, Lakshmi Narendra; Bezbaruah, Babul Kumar

    2014-10-01

    The highly conserved abundant nuclear protein poly(ADP-ribose)polymerase1 (PARP1) functions at the center of cellular stress response and is mainly implied in DNA damage repair mechanism. Apart from its involvement in DNA damage repair, it does sway multiple vital cellular processes such as cell death pathways, cell aging, insulator function, chromatin modification, transcription and mitotic apparatus function. Since brain is the principal organ vulnerable to oxidative stress and inflammatory responses, upon stress encounters robust DNA damage can occur and intense PARP1 activation may result that will lead to various CNS diseases. In the context of soaring interest towards PARP1 as a therapeutic target for newer pharmacological interventions, here in the present review, we are attempting to give a silhouette of the role of PARP1 in the neurological diseases and the potential of its inhibitors to enter clinical translation, along with its structural and functional aspects. PMID:25049175

  6. MicroRNA-7a/b Protects against Cardiac Myocyte Injury in Ischemia/Reperfusion by Targeting Poly(ADP-Ribose) Polymerase

    PubMed Central

    Li, Bin; Li, Rui; Zhang, Chun; Bian, Hong-jun; Wang, Fu; Xiao, Jie; Liu, Shan-wen; Yi, Wei; Zhang, Ming-xiang; Wang, Shuang-xi; Zhang, Yun; Su, Guo-hai; Ji, Xiao-ping

    2014-01-01

    Objectives MicroRNA-7 (miR-7) is highly connected to cancerous cell proliferation and metastasis. It is also involved in myocardial ischemia-reperfusion (I/R) injury and is upregulated in cardiomyocyte under simulated I/R (SI/R). We aimed to investigate the role of miR-7 during myocardial I/R injury in vitro and in vivo and a possible gene target. Methods and Results Real-time PCR revealed that miR-7a/b expression was upregulated in H9c2 cells after SI/R. Flow cytometry showed SI/R-induced cell apoptosis was decreased with miR-7a/b mimic transfection but increased with miR-7a/b inhibitor in H9c2 cells. In a rat cardiac I/R injury model, infarct size determination and TUNEL assay revealed that miR-7a/b mimic decreased but miR-7a/b inhibitor increased cardiac infarct size and cardiomyocyte apoptosis as compared with controls. We previously identified an important gene connected with cell apoptosis -- poly(ADP-ribose) polymerase (PARP) -- as a candidate target for miR-7a/b and verified the target by luciferase reporter activity assay and western blot analysis. Conclusions miR-7a/b is sensitive to I/R injury and protects myocardial cells against I/R-induced apoptosis by negatively regulating PARP expression in vivo and in vitro. miR-7a/b may provide a new therapeutic approach for treatment of myocardial I/R injury. Poly(ADP-ribose) polymerase. PMID:24594984

  7. TCDD-inducible poly-ADP-ribose polymerase (TIPARP/PARP7) mono-ADP-ribosylates and co-activates liver X receptors.

    PubMed

    Bindesbøll, Christian; Tan, Susanna; Bott, Debbie; Cho, Tiffany; Tamblyn, Laura; MacPherson, Laura; Grønning-Wang, Line; Nebb, Hilde Irene; Matthews, Jason

    2016-04-01

    Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and inTiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced inTiparp(-/-)mice compared withTiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1. PMID:26814197

  8. Zinc carnosine protects against hydrogen peroxide-induced DNA damage in WIL2-NS lymphoblastoid cell line independent of poly (ADP-Ribose) polymerase expression.

    PubMed

    Ooi, Theng Choon; Mohammad, Nur Hafiza; Sharif, Razinah

    2014-12-01

    The aim of this study is to investigate the ability of zinc carnosine to protect the human lymphoblastoid (WIL2-NS) cell line from hydrogen peroxide-induced DNA damage. Cells were cultured with medium containing zinc carnosine at the concentrations of 0.4, 4, 16 and 32 μM for 9 days prior to treatment with 30 μM of hydrogen peroxide (30 min). Zinc carnosine at the concentration 16 μM was optimal in protecting cells from hydrogen peroxide-induced cytotoxicity and gave the lowest percentage of apoptotic and necrotic cells. Results showed that zinc carnosine was able to induce glutathione production and protect cells from hydrogen peroxide-induced oxidative stress at all concentration and the highest protection was observed at 32-μM zinc carnosine culture. Cytokinesis-block micronucleus cytome assay showed that cells cultured with 4-32 μM of zinc carnosine showed significant reduction in micronuclei formation, nucleoplasmic bridges and nuclear bud frequencies (p < 0.05), suggesting that these concentrations maybe optimal in protecting cells from hydrogen peroxide-induced DNA damage. However, after being challenged with hydrogen peroxide, no increase in poly(ADP-ribose) polymerase expression was observed. Thus, results from this study demonstrate that zinc carnosines possess antioxidant properties and are able to reduce hydrogen peroxide-induced DNA damage in vitro independent of poly(ADP-ribose) polymerase. Further studies are warranted to understand the mechanism of protection of zinc carnosine against hydrogen peroxide-induced damage. PMID:25326781

  9. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

    PubMed Central

    Slade, Dea; Dunstan, Mark S.; Barkauskaite, Eva; Weston, Ria; Lafite, Pierre; Dixon, Neil; Ahel, Marijan; Leys, David; Ahel, Ivan

    2011-01-01

    Posttranslational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis1. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesised from NAD by poly(ADP-ribose) polymerases (PARPs)1,2. Poly(ADP-ribose) glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death3,4. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that exhibits all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macro domain family5,6. High resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. Our insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation link to human disease. PMID:21892188

  10. Overview on poly(ADP-ribose) immuno-biomedicine and future prospects.

    PubMed

    Kanai, Yoshiyuki

    2016-01-01

    Poly(ADP-ribose), identified in 1966 independently by three groups Strassbourg, Kyoto and Tokyo, is synthesized by poly(ADP-ribose) polymerases (PARP) from NAD(+) as a substrate in the presence of Mg(2+). The structure was unique in that it has ribose-ribose linkage. In the early-1970s, however, its function in vivo/in vitro was still controversial and the antibody against it was desired to help clear its significance. Thereupon, the author tried to produce antibody against poly(ADP-ribose) in rabbits and succeeded in it for the first time in the world. Eventually, this success has led to the following two groundbreaking papers in Nature: "Naturally-occurring antibody against poly(ADP-ribose) in patients with autoimmune disease SLE", and "Induction of anti-poly(ADP-ribose) antibody by immunization with synthetic double-stranded RNA, poly(A)·poly(U)".On the way to the publication of the first paper, a reviewer gave me a friendly comment that there is "heteroclitic" fashion as a mechanism of the production of natural antibody. This comment was really a God-send for me, and became a train of power for publication of another paper, as described above. Accordingly, I thought this, I would say, episode is worth describing herein. Because of its importance in biomedical phenomena, a certain number of articles related to "heteroclitic" have become to be introduced in this review, although they were not always directly related to immuno-biological works on poly(ADP-ribose). Also, I tried to speculate on the future prospects of poly(ADP-ribose), product of PARP, as an immuno-regulatory molecule, including either induced or naturally-occurring antibodies, in view of "heteroclitic". PMID:27477457

  11. Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    PubMed Central

    Walko, Thomas D; Di Caro, Valentina; Piganelli, Jon; Billiar, Timothy R; Clark, Robert SB; Aneja, Rajesh K

    2014-01-01

    Pathophysiological conditions that lead to the release of the prototypic damage-associated molecular pattern molecule high mobility group box 1 (HMGB1) also result in activation of poly(ADP-ribose) polymerase 1 (PARP1; now known as ADP-ribosyl transferase 1 [ARTD1]). Persistent activation of PARP1 promotes energy failure and cell death. The role of poly(ADP-ribosyl)ation in HMGB1 release has been explored previously; however, PARP1 is a versatile enzyme and performs several other functions including cross-talk with another nicotinamide adenine dinucleotide- (NAD+) dependent member of the Class III histone deacetylases (HDACs), sirtuin-1 (SIRT1). Previously, it has been shown that the hyperacetylation of HMGB1 is a seminal event prior to its secretion, a process that also is dependent on HDACs. Therefore, in this study, we seek to determine if PARP1 inhibition alters LPS-mediated HMGB1 hyperacetylation and subsequent secretion due to its effect on SIRT1. We demonstrate in an in vitro model that LPS treatment leads to hyperacetylated HMGB1 with concomitant reduction in nuclear HDAC activity. Treatment with PARP1 inhibitors mitigates the LPS-mediated reduction in nuclear HDAC activity and decreases HMGB1 acetylation. By utilizing an NAD+-based mechanism, PARP1 inhibition increases the activity of SIRT1. Consequently, there is an increased nuclear retention and decreased extracellular secretion of HMGB1. We also demonstrate that PARP1 physically interacts with SIRT1. Further confirmation of this data was obtained in a murine model of sepsis, that is, administration of PJ-34, a specific PARP1 inhibitor, led to decreased serum HMGB1 concentrations in mice subjected to cecal ligation and puncture (CLP) as compared with untreated mice. In conclusion, our study provides new insights in understanding the molecular mechanisms of HMGB1 secretion in sepsis. PMID:25517228

  12. Poly(ADP-ribose) Polymerase 1 Interacts with Nuclear Respiratory Factor 1 (NRF-1) and Plays a Role in NRF-1 Transcriptional Regulation*S⃞

    PubMed Central

    Hossain, Mohammad B.; Ji, Ping; Anish, Ramakrishnan; Jacobson, Raymond H.; Takada, Shinako

    2009-01-01

    Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional activators for nuclear-coded genes involved in mitochondrial biogenesis and function as well as for many housekeeping genes. A transcriptional co-activator PGC-1 and its related family member PRC have previously been shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1 can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and co-purify the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex. Our in vitro binding experiments show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of PARP-1, which contains two Zinc fingers and the auto-modification domain, are responsible for the interaction, and that this interaction occurs with or without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a complex with PARP-1, suggesting that NRF-1 can recruit the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex to the promoter. PARP-1 can also PARylate the DNA-binding domain of NRF-1 and negatively regulate NRF-1·PARP-1 interaction. Transient transfection and chromatin immunoprecipitation experiments suggest that PARP-1 plays a role during transcriptional activation by NRF-1. Our finding identifies a new aspect of transcriptional regulation used by NRF-1. PMID:19181665

  13. Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity

    SciTech Connect

    Nagy, Edit; Department of Cardiology, Karolinska University Hospital, Stockholm ; Caidahl, Kenneth; Department of Clinical Physiology, Karolinska University Hospital, Stockholm ; Franco-Cereceda, Anders; Department of Throracic Surgery, Karolinska University Hospital, Stockholm ; Baeck, Magnus; Department of Cardiology, Karolinska University Hospital, Stockholm

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. Black-Right-Pointing-Pointer We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. Black-Right-Pointing-Pointer Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. Black-Right-Pointing-Pointer The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. Black-Right-Pointing-Pointer Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C{sub 4} (LTC{sub 4}) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = -0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = -0.498; P = 0.0298) only in tricuspid aortic valves

  14. Deficiency in Poly(ADP-ribose) Polymerase-1 (PARP-1) Accelerates Aging and Spontaneous Carcinogenesis in Mice

    PubMed Central

    Piskunova, Tatiana S.; Yurova, Maria N.; Ovsyannikov, Anton I.; Semenchenko, Anna V.; Zabezhinski, Mark A.; Popovich, Irina G.; Wang, Zhao-Qi; Anisimov, Vladimir N.

    2008-01-01

    Genetic and biochemical studies have shown that PARP-1 and poly(ADP-ribosyl)ation play an important role in DNA repair, genomic stability, cell death, inflammation, telomere maintenance, and suppressing tumorigenesis, suggesting that the homeostasis of poly(ADP-ribosyl)ation and PARP-1 may also play an important role in aging. Here we show that PARP-1−/− mice exhibit a reduction of life span and a significant increase of population aging rate. Analysis of noninvasive parameters, including body weight gain, body temperature, estrous function, behavior, and a number of biochemical indices suggests the acceleration of biological aging in PARP-1−/− mice. The incidence of spontaneous tumors in both PARP-1−/− and PARP-1+/+ groups is similar; however, malignant tumors including uterine tumors, lung adenocarcinomas and hepatocellular carcinomas, develop at a significantly higher frequency in PARP-1−/− mice than PARP-1+/+ mice (72% and 49%, resp.; P < .05). In addition, spontaneous tumors appear earlier in PARP-1−/− mice compared to the wild type group. Histopathological studies revealed a wide spectrum of tumors in uterus, ovaries, liver, lungs, mammary gland, soft tissues, and lymphoid organs in both groups of the mice. These results demonstrate that inactivation of DNA repair gene PARP-1 in mice leads to acceleration of aging, shortened life span, and increased spontaneous carcinogenesis. PMID:19415146

  15. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kappaB-dependent signaling.

    PubMed

    Castri, Paola; Lee, Yang-Ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

    2014-03-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kappaB activity when expressed in neurons. PARP-1 cleavage generates a 24 kDa (PARP-1(24)) and an 89 kDa fragment (PARP-1(89)). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1(UNCL)) or of PARP-1(24) conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-1(89) was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-1(24) was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kappaB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regard to induction of NF-kappaB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-1(89) construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kappaB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-1(24) decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of INOS transcript) and lower protein expression of Bcl-xL Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of "ischemia". PMID:24333653

  16. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

    PubMed Central

    Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

  17. Rationale for Poly(ADP-ribose) Polymerase (PARP) Inhibitors in Combination Therapy with Camptothecins or Temozolomide Based on PARP Trapping versus Catalytic Inhibition

    PubMed Central

    Murai, Junko; Zhang, Yiping; Morris, Joel; Ji, Jiuping; Takeda, Shunichi; Doroshow, James H.

    2014-01-01

    We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD+-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors. PMID:24650937

  18. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    SciTech Connect

    Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

    2014-08-29

    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

  19. Protective actions of PJ34, a poly(ADP-ribose)polymerase inhibitor, on the blood-brain barrier after traumatic brain injury in mice.

    PubMed

    Tao, X; Chen, X; Hao, S; Hou, Z; Lu, T; Sun, M; Liu, B

    2015-04-16

    Poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays an important role in traumatic brain injury (TBI). The objective of this study was to investigate whether PARP activation participated in the blood-brain barrier (BBB) disruption and edema formation in a mouse model of controlled cortical impact (CCI). N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) (10 mg/kg), a selective PARP inhibitor, was administered intraperitoneally at 5 min and 8 h after experimental CCI. After 6 h and 24 h of CCI, the permeability of the cortical BBB was determined after Evans Blue administration. The water content of the brain was also measured. Treatment with PJ34 markedly attenuated the permeability of the BBB and decreased the brain edema at 6 h and 24 h after CCI. Our data showed the up-regulation of nuclear factor-κB in cytosolic fractions and nuclear fractions in the injured cortex, and these changes were reversed by PJ34. Moreover, PJ34 significantly lessened the activities of myeloperoxidase and the levels of matrix metalloproteinase-9, enhanced the levels of occludin, laminin, collagen IV and integrin β1, reduced neurological deficits, decreased the contusion volume, and attenuated the necrotic and apoptotic neuronal cell death. These data suggest the protective effects of PJ34 on BBB integrity and cell death during acute TBI. PMID:25668593

  20. Induction of Poly(ADP-ribose) Polymerase in Mouse Bone Marrow Stromal Cells Exposed to 900 MHz Radiofrequency Fields: Preliminary Observations

    PubMed Central

    He, Qina; Sun, Yulong; Zong, Lin; Tong, Jian; Cao, Yi

    2016-01-01

    Background. Several investigators have reported increased levels of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme which plays an important role in the repair of damaged DNA, in cells exposed to extremely low dose ionizing radiation which does not cause measurable DNA damage. Objective. To examine whether exposure of the cells to nonionizing radiofrequency fields (RF) is capable of increasing messenger RNA of PARP-1 and its protein levels in mouse bone marrow stromal cells (BMSCs). Methods. BMSCs were exposed to 900 MHz RF at 120 μW/cm2 power intensity for 3 hours/day for 5 days. PARP-1 mRNA and its protein levels were examined at 0, 0.5, 1, 2, 4, 6, 8, and 10 hours after exposure using RT-PCR and Western blot analyses. Sham-exposed (SH) cells and those exposed to ionizing radiation were used as unexposed and positive control cells. Results. BMSCs exposed to RF showed significantly increased expression of PARP-1 mRNA and its protein levels after exposure to RF while such changes were not observed in SH-exposed cells. Conclusion. Nonionizing RF exposure is capable of inducing PARP-1. PMID:27190989

  1. Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    PubMed Central

    Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

    2015-01-01

    Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

  2. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes.

    PubMed

    Cuvillier, O; Rosenthal, D S; Smulson, M E; Spiegel, S

    1998-01-30

    Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7. PMID:9446602

  3. Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota

    2016-07-01

    Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression. PMID:27155387

  4. Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.

    PubMed

    Liu, Yie; Snow, Bryan E; Kickhoefer, Valerie A; Erdmann, Natalie; Zhou, Wen; Wakeham, Andrew; Gomez, Marla; Rome, Leonard H; Harrington, Lea

    2004-06-01

    Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo. PMID:15169895

  5. Differential regulation of activator protein-1 and heat shock factor-1 in myocardial ischemia and reperfusion injury: role of poly(ADP-ribose) polymerase-1.

    PubMed

    Zingarelli, Basilia; Hake, Paul W; O'Connor, Michael; Denenberg, Alvin; Wong, Hector R; Kong, Sue; Aronow, Bruce J

    2004-04-01

    Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated in response to DNA strand breaks, has been implicated in cell dysfunction in myocardial reperfusion injury. PARP-1 has also been shown to participate in transcription and regulation of gene expression. In this study, we investigated the role of PARP-1 on the signal transduction pathway of activator protein-1 (AP-1) and heat shock factor-1 (HSF-1) in myocardial reperfusion injury. Mice genetically deficient of PARP-1 (PARP-1(-/-) mice) exhibited a significant reduction of myocardial damage after occlusion and reperfusion of the left anterior descending branch of the coronary artery compared with their wild-type littermates. This cardioprotection was associated with a reduction of the phosphorylative activity of JNK and, subsequently, reduction of the DNA binding of the signal transduction factor AP-1. On the contrary, in PARP-1(-/-) mice, DNA binding of HSF-1 was enhanced and was associated with a significant increase of the cardioprotective heat shock protein (HSP)70 compared with wild-type mice. Microarray analysis revealed that expression of several AP-1-dependent genes of proinflammatory mediators and HSPs was altered in PARP-1(-/-) mice. The data indicate that PARP-1 may exert a pathological role in reperfusion injury by functioning as an enhancing factor of AP-1 activation and as a repressing factor of HSF-1 activation and HSP70 expression. PMID:14670820

  6. Hydrogen-rich saline reduces cell death through inhibition of DNA oxidative stress and overactivation of poly (ADP-ribose) polymerase-1 in retinal ischemia-reperfusion injury.

    PubMed

    Liu, Hongwei; Hua, Ning; Xie, Keliang; Zhao, Tingting; Yu, Yonghao

    2015-08-01

    Overactivation of poly (ADP-ribose) polymerase 1 (PARP-1), as a result of sustained DNA oxidation in ischemia-reperfusion injury, triggers programmed cell necrosis and apoptosis. The present study was conducted to demonstrate whether hydrogen-rich saline (HRS) has a neuroprotective effect on retinal ischemia reperfusion (RIR) injury through inhibition of PARP-1 activation. RIR was induced by transient elevation of intraocular pressure in rats. HRS (5 ml/kg) was administered peritoneally every day from the beginning of reperfusion in RIR rats until the rats were sacrificed. Retinal damage and cell death was determined using hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. DNA oxidative stress was evaluated by immunofluorescence staining of 8-hydroxy-2-deoxyguanosine. In addition, the expression of PARP-1 and caspase-3 was investigated by western blot analysis and/or immunohistochemical staining. The results demonstrated that HRS administration improved morphological alterations and reduced apoptosis following RIR injury. Furthermore, the present study found that HRS alleviated DNA oxidation and PARP-1 overactivation in RIR rats. HRS can protect RIR injury by inhibition of PARP-1, which may be involved in DNA oxidative stress and caspase-3-mediated apoptosis. PMID:25954991

  7. Hydrogen-rich saline reduces cell death through inhibition of DNA oxidative stress and overactivation of poly (ADP-ribose) polymerase-1 in retinal ischemia-reperfusion injury

    PubMed Central

    LIU, HONGWEI; HUA, NING; XIE, KELIANG; ZHAO, TINGTING; YU, YONGHAO

    2015-01-01

    Overactivation of poly (ADP-ribose) polymerase 1 (PARP-1), as a result of sustained DNA oxidation in ischemia-reperfusion injury, triggers programmed cell necrosis and apoptosis. The present study was conducted to demonstrate whether hydrogen-rich saline (HRS) has a neuroprotective effect on retinal ischemia reperfusion (RIR) injury through inhibition of PARP-1 activation. RIR was induced by transient elevation of intraocular pressure in rats. HRS (5 ml/kg) was administered peritoneally every day from the beginning of reperfusion in RIR rats until the rats were sacrificed. Retinal damage and cell death was determined using hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. DNA oxidative stress was evaluated by immunofluorescence staining of 8-hydroxy-2-deoxyguanosine. In addition, the expression of PARP-1 and caspase-3 was investigated by western blot analysis and/or immunohistochemical staining. The results demonstrated that HRS administration improved morphological alterations and reduced apoptosis following RIR injury. Furthermore, the present study found that HRS alleviated DNA oxidation and PARP-1 overactivation in RIR rats. HRS can protect RIR injury by inhibition of PARP-1, which may be involved in DNA oxidative stress and caspase-3-mediated apoptosis. PMID:25954991

  8. Targeting Poly (ADP-Ribose) Polymerase and the c-Myb-TopBP1-ATR-Chk1 Signaling Pathway in Castration-Resistant Prostate Cancer

    PubMed Central

    Li, Likun; Chang, Wenjun; Yang, Guang; Ren, Chengzhen; Park, Sanghee; Karantanos, Theodoros; Karanika, Styliani; Wang, Jianxiang; Yin, Jianhua; Shah, Parantu K.; Takahiro, Hirayama; Dobashi, Masato; Zhang, Wenling; Efstathiou, Eleni; Maity, Sankar N.; Aparicio, Ana M.; Tapia, Elsa M Li Ning; Troncoso, Patricia; Broom, Bradley; Xiao, Lianchun; Lee, Hyun-Sung; Lee, Ju-Seog; Corn, Paul G.; Navone, Nora; Thompson, Timothy C.

    2014-01-01

    Androgen deprivation is the standard systemic treatment for advanced prostate cancer (PCa), but most patients ultimately develop castration-resistance. We show here that MYB is transcriptionally activated by androgen deprivation or impairment of androgen receptor (AR) signaling. MYB gene silencing significantly inhibited PCa growth in vitro and in vivo. Microarray data revealed that c-Myb shares a substantial subset of DNA damage response (DDR) target genes with AR, suggesting that c-Myb may replace AR for the dominant role in the regulation of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures comprising AR, MYB, and their common DDR target genes are significantly correlated with metastasis, castration-resistance, recurrence, and shorter overall survival in PCa patients. We demonstrated in vitro that silencing of MYB, BRCA1 or TOPBP1 synergized with poly (ADP-ribose) polymerase (PARP) inhibitor olaparib (OLA) to increase cytotoxicity to PCa cells. We further demonstrated that targeting the c-Myb-TopBP1-ATR-Chk1 pathway by using the Chk1 inhibitor AZD7762 synergizes with OLA to increase PCa cytotoxicity. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the c-Myb-TopBP1-ATR-Chk1 pathway. PMID:24847116

  9. Differential effects of poly(ADP-ribose) polymerase inhibition on DNA break repair in human cells are revealed with Epstein-Barr virus.

    PubMed

    Ma, Wenjian; Halweg, Christopher J; Menendez, Daniel; Resnick, Michael A

    2012-04-24

    Poly(ADP-ribose) polymerase (PARP) inhibitors can generate synthetic lethality in cancer cells defective in homologous recombination. However, the mechanism(s) by which they affect DNA repair has not been established. Here we directly determined the effects of PARP inhibition and PARP1 depletion on the repair of ionizing radiation-induced single- and double-strand breaks (SSBs and DSBs) in human lymphoid cell lines. To do this, we developed an in vivo repair assay based on large endogenous Epstein-Barr virus (EBV) circular episomes. The EBV break assay provides the opportunity to assess quantitatively and simultaneously the induction and repair of SSBs and DSBs in human cells. Repair was efficient in G1 and G2 cells and was not dependent on functional p53. shRNA-mediated knockdown of PARP1 demonstrated that the PARP1 protein was not essential for SSB repair. Among 10 widely used PARP inhibitors, none affected DSB repair, although an inhibitor of DNA-dependent protein kinase was highly effective at reducing DSB repair. Only Olaparib and Iniparib, which are in clinical cancer therapy trials, as well as 4-AN inhibited SSB repair. However, a decrease in PARP1 expression reversed the ability of Iniparib to reduce SSB repair. Because Iniparib disrupts PARP1-DNA binding, the mechanism of inhibition does not appear to involve trapping PARP at SSBs. PMID:22493268

  10. Rationale for poly(ADP-ribose) polymerase (PARP) inhibitors in combination therapy with camptothecins or temozolomide based on PARP trapping versus catalytic inhibition.

    PubMed

    Murai, Junko; Zhang, Yiping; Morris, Joel; Ji, Jiuping; Takeda, Shunichi; Doroshow, James H; Pommier, Yves

    2014-06-01

    We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD(+)-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors. PMID:24650937

  11. Neuroprotective effects of a novel water-soluble poly(ADP-ribose) polymerase-1 inhibitor, MP-124, in in vitro and in vivo models of cerebral ischemia.

    PubMed

    Egi, Yasuhiro; Matsuura, Shigeru; Maruyama, Tomoyuki; Fujio, Masakazu; Yuki, Satoshi; Akira, Toshiaki

    2011-05-10

    Cerebral ischemia induces excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), leading to neuronal cell death and the development of post-ischemic dysfunction. Blockade of PARP-related signals during cerebral ischemia has become a focus of interest as a new therapeutic approach for acute stroke treatment. The purpose of the present study was to examine the pharmacological profiles of MP-124, a novel water-soluble PARP-1 inhibitor, and its neuroprotective effects on ischemic injury in vitro and in vivo. MP-124 demonstrated competitive inhibition of the PARP-1 activity of human recombinant PARP-1 enzyme (Ki=16.5nmol/L). In P388D(1) cells, MP-124 inhibited the LDH leakage induced by H(2)O(2) in a concentration-dependent manner. (IC(50)=20.8nmol/L). In rat primary cortical neurons, MP-124 also inhibited the NAD depletion and polymerized ADP-ribose formation induced by H(2)O(2) exposure. Moreover, we investigated the neuroprotective effects of MP-124 in rat permanent and transient stroke models. In the rat permanent middle cerebral artery occlusion (MCAO) model, MP-124 was administered intravenously for 24h from 5min after the onset of MCAO. MP-124 (1, 3 and 10mg/kg/h) significantly inhibited the cerebral infarction in a dose-dependent manner (18, 42 and 48%). In rat transient MCAO model, MP-124 was administered intravenously from 30min after the onset of MCAO. MP-124 (3 and 10mg/kg/h) significantly reduced the infarct volume (53% and 50%). The present findings suggest that MP-124 acts as a potent neuroprotective agent in focal ischemia and its actions can be attributed to a reduction in NAD depletion and PAR formation. PMID:21420942

  12. Glucagon-Like Peptide 1 Protects against Hyperglycemic-Induced Endothelial-to-Mesenchymal Transition and Improves Myocardial Dysfunction by Suppressing Poly(ADP-Ribose) Polymerase 1 Activity

    PubMed Central

    Yan, Fei; Zhang, Guang-hao; Feng, Min; Zhang, Wei; Zhang, Jia-ning; Dong, Wen-qian; Zhang, Cheng; Zhang, Yun; Chen, Li; Zhang, Ming-Xiang

    2015-01-01

    Under high glucose conditions, endothelial cells respond by acquiring fibroblast characteristics, that is, endothelial-to-mesenchymal transition (EndMT), contributing to diabetic cardiac fibrosis. Glucagon-like peptide-1 (GLP-1) has cardioprotective properties independent of its glucose-lowering effect. However, the potential mechanism has not been fully clarified. Here we investigated whether GLP-1 inhibits myocardial EndMT in diabetic mice and whether this is mediated by suppressing poly(ADP-ribose) polymerase 1 (PARP-1). Streptozotocin diabetic C57BL/6 mice were treated with or without GLP-1 analog (24 nmol/kg daily) for 24 wks. Transthoracic echocardiography was performed to assess cardiac function. Human aortic endothelial cells (HAECs) were cultured in normal glucose (NG) (5.5 mmol/L) or high glucose (HG) (30 mmol/L) medium with or without GLP-1analog. Immunofluorescent staining and Western blot were performed to evaluate EndMT and PARP-1 activity. Diabetes mellitus attenuated cardiac function and increased cardiac fibrosis. Treatment with the GLP-1 analog improved diabetes mellitus–related cardiac dysfunction and cardiac fibrosis. Immunofluorescence staining revealed that hyperglycemia markedly increased the percentage of von Willebrand factor (vWF)+/alpha smooth muscle actin (α-SMA)+ cells in total α-SMA+ cells in diabetic hearts compared with controls, which was attenuated by GLP-1 analog treatment. In cultured HAECs, immunofluorescent staining and Western blot also showed that both GLP-1 analog and PARP-1 gene silencing could inhibit the HG-induced EndMT. In addition, GLP-1 analog could attenuate PARP-1 activation by decreasing the level of reactive oxygen species (ROS). Therefore, GLP-1 treatment could protect against the hyperglycemia-induced EndMT and myocardial dysfunction. This effect is mediated, at least partially, by suppressing PARP-1 activation. PMID:25715248

  13. p38γ Mitogen-Activated Protein Kinase Contributes to Oncogenic Properties Maintenance and Resistance to Poly (ADP-Ribose)-Polymerase-1 Inhibition in Breast Cancer12

    PubMed Central

    Meng, Fanyan; Zhang, Haijun; Liu, Gang; Kreike, Bas; Chen, Wei; Sethi, Seema; Miller, Fred R; Wu, Guojun

    2011-01-01

    p38γ MAPK, one of the four members of p38 mitogen-activated protein kinases (MAPKs), has previously been shown to harbor oncogenic functions. However, the biologic function of p38γ MAPK in breast cancer has not been well defined. In this study, we have shown that p38γ MAPK is overexpressed in highly metastatic human and mouse breast cancer cell lines and p38γ MAPK expression is preferentially associated with basal-like and metastatic phenotypes of breast tumor samples. Ectopic expression of p38γ MAPK did not lead to an increase in oncogenic properties in vitro in most tested mammary epithelial cells. However, knockdown of p38γ MAPK expression resulted in a dramatic decrease in cell proliferation, colony formation, cell migration, invasion in vitro and significant retardation of tumorigenesis, and long-distance metastasis to the lungs in vivo. Moreover, knockdown of p38γ MAPK triggered the activation of AKT signaling. Inhibition of this feedback loop with various PI3K/AKT signaling inhibitors facilitated the effect of targeting p38γ MAPK. We further found that overexpression of p38γ MAPK did not promote cell resistance to chemotherapeutic agents doxorubicin and paclitaxel but significantly increased cell resistance to PJ-34, a DNA damage agent poly (ADP-ribose)-polymerase-1 (PARP) inhibitor in vitro and in vivo. Finally, we identified that p38γ MAPK overexpression led to marked cell cycle arrest in G2/M phase. Our study for the first time clearly demonstrates that p38γ MAPK is a promising target for the design of targeted therapies for basal-like breast cancer with metastatic characteristics and for overcoming potential resistance against the PARP inhibitor. PMID:21532888

  14. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

    PubMed Central

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2′-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2′-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs. PMID:26366077

  15. The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

    PubMed

    Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

    2016-06-10

    Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. PMID:27068743

  16. Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption.

    PubMed

    Halappanavar, Sabina S; Shah, Girish M

    2004-03-01

    Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints. PMID:14726664

  17. Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

    SciTech Connect

    Mitchell, Jody; Smith, Graeme; Curtin, Nicola J.

    2009-12-01

    Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.

  18. Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer

    PubMed Central

    Owonikoko, Taofeek K; Zhang, Guojing; Deng, Xingming; Rossi, Michael R; Switchenko, Jeffrey M; Doho, Gregory H; Chen, Zhengjia; Kim, Sungjin; Strychor, Sandy; Christner, Susan M; Beumer, Jan; Li, Chunyang; Yue, Ping; Chen, Alice; Sica, Gabriel L; Ramalingam, Suresh S; Kowalski, Jeanne; Khuri, Fadlo R; Sun, Shi-Yong

    2014-01-01

    Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single-agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell-specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile. PMID:25124282

  19. A Role for Poly(ADP-ribose) Polymerase in the Transcriptional Regulation of the Melanoma Growth Stimulatory Activity (CXCL1) Gene Expression*

    PubMed Central

    Nirodi, Chaitanya; NagDas, Subir; Gygi, Steven P.; Olson, Gary; Aebersold, Ruedi; Richmond, Ann

    2012-01-01

    The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-κB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides −94 to −78), which lies immediately upstream to the nuclear factor κB (NF-κB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds a 115-kDa protein. In this study, the IUR element has been further characterized. We show here that proximity of the IUR element to the adjacent NF-κB element is critical to its function as a positive regulatory element. Using binding site oligonucleotide affinity chromatography, we have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy and amino acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-aminobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA. The data point to the possibility that PARP may be a coactivator of CXCL1 transcription. PMID:11112786

  20. The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

    PubMed Central

    Rom, Slava; Reichenbach, Nancy L.; Dykstra, Holly; Persidsky, Yuri

    2015-01-01

    Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60–80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85–95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection. PMID:26379653

  1. Modulation of the poly (ADP-ribose) polymerase inhibitor response and DNA recombination in breast cancer cells by drugs affecting endogenous wild-type p53.

    PubMed

    Ireno, Ivanildce Cristiane; Wiehe, Rahel Stephanie; Stahl, Andreea Iulia; Hampp, Stephanie; Aydin, Sevtap; Troester, Melissa A; Selivanova, Galina; Wiesmüller, Lisa

    2014-10-01

    Synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) and homologous recombination (HR) repair pathways have been exploited for the development of novel mono- and combination cancer therapies. The tumor suppressor p53 was demonstrated to exhibit indirect and direct regulatory activities in DNA repair, particularly in DNA double-strand break (DSB)-induced and replication-associated HR. In this study, we tested a potential influence of the p53 status on the response to PARP inhibition, which is known to cause replication stress. Silencing endogenous or inducibly expressing p53 we found a protective effect of p53 on PARP inhibitor (PARPi)-mediated cytotoxicities. This effect was specific for wild-type versus mutant p53 and observed in cancer but not in non-transformed cell lines. Enhanced cytotoxicities after treatment with the p53-inhibitory drug Pifithrinα further supported p53-mediated resistance to PARP inhibition. Surprisingly, we equally observed increased PARPi sensitivity in the presence of the p53-activating compound Nutlin-3. As a common denominator, both drug responses correlated with decreased HR activities: Pifithrinα downregulated spontaneous HR resulting in damage accumulation. Nutlin-3 induced a decrease of DSB-induced HR, which was accompanied by a severe drop in RAD51 protein levels. Thus, we revealed a novel link between PARPi responsiveness and p53-controlled HR activities. These data expand the concept of cell and stress type-dependent healer and killer functions of wild-type p53 in response to cancer therapeutic treatment. Our findings have implications for the individualized design of cancer therapies using PARPi and the potentially combined use of p53-modulatory drugs. PMID:25085902

  2. Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase.

    PubMed

    Somyajit, Kumar; Mishra, Anup; Jameei, Aida; Nagaraju, Ganesh

    2015-01-01

    Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a 'synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G2/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity 'synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a 'synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes. PMID:25292178

  3. Activity of CEP-9722, a poly (ADP-ribose) polymerase inhibitor, in urothelial carcinoma correlates inversely with homologous recombination repair response to DNA damage.

    PubMed

    Jian, Weiguo; Xu, Hua-Guo; Chen, Jianfeng; Xu, Zhi-Xiang; Levitt, Jonathan M; Stanley, Jennifer A; Yang, Eddy S; Lerner, Seth P; Sonpavde, Guru

    2014-09-01

    As loss of DNA-repair proteins is common in urothelial carcinoma (UC), a rationale can be made to evaluate the activity of poly (ADP-ribose) polymerase (PARP) inhibitors to exploit synthetic lethality. We aimed to preclinically evaluate a PARP inhibitor, CEP-9722, and its active metabolite, CEP-8983, in UC. The activity of CEP-8983 was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay against human UC cell lines. Flow cytometry, COMET assay, and western blot were performed to assess apoptosis, DNA damage, and DNA-repair proteins, respectively. RT4 xenografts received placebo or CEP-9722 (100 or 200 mg/kg/day) orally. Xenografts were subjected to immunohistochemistry for apoptosis [cleaved caspase (cc)-3] and angiogenesis (CD31). CEP-8983 (1 μmol/l) reduced the viability of RT4 and T24 cells by 20%, but did not reduce the viability of 5637 and TCC-SUP cells. Apoptosis and necrosis occurred in 9.7 and 9.1% of RT4 and 5637 cells, respectively. RT4 cells showed greater DNA damage compared with 5637 cells. Increased DNA damage occurred with combination versus CEP-8983 or cisplatin alone in RT4 and 5637 cells. T24 and RT4 showed the least RAD51 foci 8 h following radiation, whereas TCC-SUP and 5637 robustly induced RAD51 foci. CEP-9722 showed dose-dependent antitumor activity in RT4 xenografts; 200 mg/kg daily was better than control (P=0.04) and 100 mg/kg was not (P=0.26). Immunohistochemistry of xenografts showed a significant increase in cc-3 and decrease in CD31 with both doses (P<0.05). Biomarker-driven evaluation of PARP inhibitors in UC is justified as the activity of CEP-9722 correlated inversely with homologous recombination repair response to DNA damage. PMID:24714082

  4. Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?

    PubMed Central

    Lechaftois, Marie; Dreano, Elise; Palmier, Bruno; Margaill, Isabelle; Marchand-Leroux, Catherine; Bachelot-Loza, Christilla; Lerouet, Dominique

    2014-01-01

    Background Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001. Methods and results ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y1 and P2Y12. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y12 pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used. Conclusions We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y12 antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment. PMID:25329809

  5. Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex.

    PubMed

    Czapski, Grzegorz A; Cakala, Magdalena; Kopczuk, Dorota; Strosznajder, Joanna B

    2004-02-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair, replication and cell cycle. However, its overactivation leads to nicotinamide adenine dinucleotide and ATP depletion and cell death. The inhibitors of PARP-1 were successfully used in the basic studies and in animal models of different diseases. For this reason, it is important to discriminate between specific and non-specific antioxidant properties of PARP-1 inhibitors. The aim of this study was to investigate the effect of PARP-1 inhibitors on the free radical level and oxidation of macromolecules and to compare their properties with the efficacy of antioxidants. Oxidative stress was induced in the brain cortex homogenate by FeCl(2) or CuSO(4) at 25 microM during 15 min incubation at 37 degrees C. PARP-1 inhibitors 3-aminobenzamide (3-AB), 1,5-dihydroxyisoquinoline (DHIQ) and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), and the antioxidants alpha-tocopherol, resveratrol and Tempol were used at 0-5 mM. Free radical contents were estimated by spin-trapping using HPLC. Lipid and protein oxidation were determined by measuring thiobarbituric acid reactive substances and carbonyl groups or using fluorescent probe TyrFluo, respectively. Our data indicate that 3-AB and DHIQ are potent hydroxyl radical scavengers and inhibitors of protein oxidation. DHIQ additionally decreases lipid peroxidation. DPQ has no antioxidant properties and seems to be a specific PARP-1 inhibitor, however, it is a water insoluble compound. Among the investigated antioxidants, the most potent was resveratrol and then alpha-tocopherol and Tempol. These results indicate that 3-A beta, benzamide and DHIQ are potent hydroxyl radical scavengers and antioxidants. These data ought to be taken into consideration when properties of these compounds as PARP inhibitors are evaluated. PMID:14746898

  6. Modulation of farnesoid X receptor results in post-translational modification of poly (ADP-ribose) polymerase 1 in the liver

    SciTech Connect

    Zhu, Yan; Li, Guodong; Dong, Yafeng; Zhou, Helen H.; Kong, Bo; Aleksunes, Lauren M.; Richardson, Jason R.; Li, Fei; Guo, Grace L.

    2013-01-15

    The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in mice leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3 month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18 months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver. -- Highlights: ► Proteomic analysis identified novel FXR targets. ► FXR modification altered post-translational modification of the Parp1 protein. ► Altered Parp1 function may contribute to mechanisms of FXR regulation of liver functions.

  7. Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo

    PubMed Central

    Liu, Feng; Jiang, Ning; Xiao, Zhi-yong; Cheng, Jun-ping; Mei, Yi-zhou; Zheng, Pan; Wang, Li; Zhang, Xiao-rui; Zhou, Xin-bo

    2016-01-01

    Early studies with first-generation poly (ADP-ribose) polymerase (PARP) inhibitors have already indicated some therapeutic potential for sulfur mustard (SM) injuries. The available novel and more potential PARP inhibitors, which are undergoing clinical trials as drugs for cancer treatment, bring it back to the centre of interest. However, the role of PARP-1 in SM-induced injury is not fully understood. In this study, we selected a high potent specific PARP inhibitor ABT-888 as an example to investigate the effect of PARP inhibitor in SM injury. The results showed that in both the mouse ear vesicant model (MEVM) and HaCaT cell model, PARP inhibitor ABT-888 can reduce cell damage induced by severe SM injury. ABT-888 significantly reduced SM induced edema and epidermal necrosis in MEVM. In the HaCaT cell model, ABT-888 can reduce SM-induced NAD+/ATP depletion and apoptosis/necrosis. Then, we studied the mechanism of PARP-1 in SM injury by knockdown of PARP-1 in HaCaT cells. Knockdown of PARP-1 protected cell viability and downregulated the apoptosis checkpoints, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP and Caspase 3 following SM-induced injury. Furthermore, the activation of AKT can inhibit autophagy via the regulation of mTOR. Our results showed that SM exposure could significantly inhibit the activation of Akt/mTOR pathway. Knockdown of PARP-1 reversed the SM-induced suppression of the Akt/mTOR pathway. In summary, the results of our study indicated that the protective effects of downregulation of PARP-1 in SM injury may be due to the regulation of apoptosis, necrosis, energy crisis and autophagy. However, it should be noticed that PARP inhibitor ABT-888 further enhanced the phosphorylation of H2AX (S139) after SM exposure, which indicated that we should be very careful in the application of PARP inhibitors in SM injury treatment because of the enhancement of DNA damage. PMID:27077006

  8. Interdependent genotoxic mechanisms of monomethylarsonous acid: Role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells

    SciTech Connect

    Wnek, Shawn M.; Kuhlman, Christopher L.; Camarillo, Jeannie M.; Medeiros, Matthew K.; Liu, Ke J.; Lau, Serrine S.; Gandolfi, A.J.

    2011-11-15

    Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA{sup III}), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA{sup III} exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA{sup III}, PARP-1 activity does not increase despite the increase in MMA{sup III}-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA{sup III} exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA{sup III} indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA{sup III}. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA{sup III} to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA{sup III} to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA{sup III} exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA{sup III}. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA{sup III}-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA

  9. A duplicated region is responsible for the poly(ADP-ribose) polymerase polymorphism, on chromosome 13, associated with a predisposition to cancer

    SciTech Connect

    Lyn, D.; Cherney, B.W.; Lupold, S.; Smulson, M. ); Lalande, M. Harvard Medical School, Boston, MA ); Berenson, J.R.; Lichtenstein, A. Veterans Administration Medical Center, Los Angeles, CA ); Bhatia, K.G. )

    1993-01-01

    The poly(ADP-ribose) polymerase (PADPRP) gene (13q33-qter) depicts a two-allele (A/B) polymorphism. In the noncancer population, the frequency of the B allele is higher among blacks than among whites. Since the incidence of multiple myeloma and prostate and lung cancer is higher in the US black population, the authors have analyzed the B-allele frequency in germ-line DNA to determine whether the PADPRP gene correlates with a polymorphic susceptibility to these diseases. For multiple myeloma and prostate cancer, an increased frequency of the B allele appeared to be striking only in black patients. In contrast, the distribution of the B allele in germ-line DNA did not differ among white patients with these diseases, when compared with the control group. An elevated B-allele frequency was also found in germ-line DNA in blacks with colon cancer. These observations suggest that the PADPRP polymorphism may provide a valid marker for a predisposition to these cancers in black individuals. To determine the genomic structure of the polymorphic PADPRP sequences, a 2.68-kb HindIII clone was isolated and sequenced from a chromosome 13-enriched library. Sequence analysis of this clone (A allele) revealed a close sequence similarity (91.8%) to PADPRP cDNA (1q42) and an absence of introns, suggesting that the gene on 13q exists as a processed pseudogene. A 193-bp conserved duplicated region within the A allele was identified as the source of the polymorphism. The nucleotide differences between the PADPRP gene on chromosome 13 and related PADPRP genes were exploited to develop oligonucleotides that can detect the difference between the A/B genotypes in a PCR. This PCR assay offers the opportunity for analyzing additional black cancer patients, to determine how the PADPRP processed pseudogene or an unidentified gene that cosegregates with the PADPRP gene might be involved with the development of malignancy. 16 refs., 6 figs., 1 tab.

  10. Extracellular poly(ADP-ribose) is a pro-inflammatory signal for macrophages

    PubMed Central

    Krukenberg, Kristin A.; Kim, Sujeong; Tan, Edwin S.; Maliga, Zoltan; Mitchison, Timothy J.

    2015-01-01

    Summary Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR), an essential post-translational modification whose function is important in many cellular processes including DNA damage signalling, cell death, and inflammation. All known PAR biology is intracellular, but we suspected it might also play a role in cell-to-cell communication during inflammation. We found that PAR activated cytokine release in human and mouse macrophages, a hallmark of innate immune activation, and determined structure-activity relationships. PAR was rapidly internalized by murine macrophages, while the monomer, ADP-ribose, was not. Inhibitors of TLR2 and TLR4 signaling blocked macrophage responses to PAR, and PAR induced TLR2 and TLR4 signaling in reporter cell lines suggesting it was recognized by these TLRs, much like bacterial pathogens. We propose that PAR acts as an extracellular “Damage Associated Molecular Pattern” (DAMP) that drives inflammatory signaling. PMID:25865309

  11. Excitotoxicity in the Lung: N-Methyl-D-Aspartate-Induced, Nitric Oxide-Dependent, Pulmonary Edema is Attenuated by Vasoactive Intestinal Peptide and by Inhibitors of Poly(ADP-Ribose) Polymerase

    NASA Astrophysics Data System (ADS)

    Said, Sami I.; Berisha, Hasan I.; Pakbaz, Hedayatollah

    1996-05-01

    Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the ``adult respiratory distress syndrome,'' and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

  12. Identification and ranking of poly (ADP-ribose) polymerase inhibitors as protectors against sulfur mustard induced decrease in cellular energy and viability in in vitro assays with human lymphocytes

    SciTech Connect

    Meier, H.L.; Kelly, S.A.

    1993-05-13

    Lymphocyte were utilized as a model for investigating HD effects on resting cells. Lymphocytes exposed to HD demonstrated a concentration dependent decrease in ATP, NAD, and viability. The decrease began in 15 minutes for ATP, 2 hours for NAD, and 6 hours for viability. All three of these HD initiated biochemical changes can be blocked by poly (ADP-ribose) polymerase inhibitors (PADPRPI). To completely inhibit HD initiated ATP, NAD, and viability decreases the PADPRPI had to be present at time 0, 1, and 4 hours respectfully. The amount of protection conferred by the PADPRPI in the viability assay decreased in a linear manner with the delay of the addition and the concentration of the inhibitor from 6-12 hours post HD exposure. There was a good correlation between IC50 to inhibit poly (ADP-ribose) polymerase and EC50 prevention of HD initiated cell death (r=O.94). Thus, three in vitro assays which can measure biochemical and pathologic changes induced by HD in G sub 0 lymphocytes have been developed. These assays have been employed to study the ability of candidate antidotes to prevent HD initiated changes. Benzamidine analogs, including the F.D.A. approved vitamin niacinamide, have been shown to be effective at inhibiting all of these changes.

  13. Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.

    PubMed Central

    Said, S I; Berisha, H I; Pakbaz, H

    1996-01-01

    Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase. Images Fig. 3 PMID:8643465

  14. Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)*

    PubMed Central

    Diani-Moore, Silvia; Zhang, Sheng; Ram, Payal; Rifkind, Arleen B.

    2013-01-01

    Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis. TCDD is a potent activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The relationship between gene activation by the AHR and TCDD toxicities is not well understood. We recently identified a pathway by which the AHR target gene TiPARP (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD+ and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a transcriptional activator of PEPCK. We report here that TCDD-induced TiPARP also targets PEPCK for ADP-ribosylation. Both cytosolic and mitochondrial forms of PEPCK were found to undergo ADP-ribosylation. Unexpectedly, AHR suppression also enhanced ADP-ribosylation and did so by a poly(ADP-ribose) polymerase-independent mechanism. This report 1) identifies ADP-ribosylation as a new posttranslational modification for PEPCK, 2) describes a pathway by which transcriptional induction of TiPARP by the AHR can lead to a downstream posttranslational change in a TCDD target protein (PEPCK), and 3) reveals that the AHR exerts complex, previously unidentified modulatory effects on ADP-ribosylation. PMID:23770670

  15. Poly(ADP-ribose): From chemical synthesis to drug design.

    PubMed

    Drenichev, Mikhail S; Mikhailov, Sergey N

    2016-08-01

    Poly(ADP-ribose) (PAR) is an important biopolymer, which is involved in various life processes such as DNA repair and replication, modulation of chromatin structure, transcription, cell differentiation, and in pathogenesis of various diseases such as cancer, diabetes, ischemia and inflammations. PAR is the most electronegative biopolymer and this property is essential for its binding with a wide range of proteins. Understanding of PAR functions in cell on molecular level requires chemical synthesis of regular PAR oligomers. Recently developed methodologies for chemical synthesis of PAR oligomers, will facilitate the study of various cellular processes, involving PAR. PMID:27318540

  16. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    PubMed Central

    Vilchez Larrea, Salomé C.; Kun, Alejandra

    2014-01-01

    Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications. PMID:25332845

  17. Poly(ADP-ribose) Signals to Mitochondrial AIF: A Key Event in Parthanatos

    PubMed Central

    Wang, Yingfei; Dawson, Valina L.; Dawson, Ted M.

    2009-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in multiple neurologic diseases by mediating caspase-independent cell death, which has recently been designated parthanatos to distinguish it from other forms of cell death such as apoptosis, necrosis and autophagy. Mitochondrial apoptosis-inducing factor (AIF) release and translocation to the nucleus is the commitment point for parthanatos. This process involves a pathogenic role of poly (ADP-ribose) (PAR) polymer. It generates in the nucleus and translocates to the mitochondria to mediate AIF release following lethal PARP-1 activation. PAR polymer itself is toxic to cells. Thus, PAR polymer signaling to mitochondrial AIF is the key event initiating the deadly crosstalk between the nucleus and the mitochondria in parthanatos. Targeting PAR-mediated AIF release could be a potential approach for the therapy of neurologic disorders. PMID:19332058

  18. Failure to degrade poly(ADP-ribose) causes increased sensitivity to cytotoxicity and early embryonic lethality

    PubMed Central

    Koh, David W.; Lawler, Ann M.; Poitras, Marc F.; Sasaki, Masayuki; Wattler, Sigrid; Nehls, Michael C.; Stöger, Tobias; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2004-01-01

    The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADP-ribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADP-ribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N′-nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress. PMID:15591342

  19. Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

    SciTech Connect

    Gao Hong; Coyle, Donna L.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Elaine L.; Wang, Zhao-Qi; Jacobson, Myron K. . E-mail: mjacobson@pharmacy.arizona.edu

    2007-03-10

    Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-{delta}2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-{delta}2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-{delta}2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.

  20. Optimization of Phenyl-Substituted Benzimidazole Carboxamide Poly(ADP-Ribose) Polymerase Inhibitors: Identification of (S)-2-(2-Fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (A-966492), a Highly Potent and Efficacious Inhibitor

    SciTech Connect

    Penning, Thomas D.; Zhu, Gui-Dong; Gong, Jianchun; Thomas, Sheela; Gandhi, Viraj B.; Liu, Xuesong; Shi, Yan; Klinghofer, Vered; Johnson, Eric F.; Park, Chang H.; Fry, Elizabeth H.; Donawho, Cherrie K.; Frost, David J.; Buchanan, Fritz G.; Bukofzer, Gail T.; Rodriguez, Luis E.; Bontcheva-Diaz, Velitchka; Bouska, Jennifer J.; Osterling, Donald J.; Olson, Amanda M.; Marsh, Kennan C.; Luo, Yan; Giranda, Vincent L.

    2010-06-21

    We have developed a series of phenylpyrrolidine- and phenylpiperidine-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase (PARP) inhibitors with excellent PARP enzyme potency as well as single-digit nanomolar cellular potency. These efforts led to the identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (22b, A-966492). Compound 22b displayed excellent potency against the PARP-1 enzyme with a K{sub i} of 1 nM and an EC{sub 50} of 1 nM in a whole cell assay. In addition, 22b is orally bioavailable across multiple species, crosses the blood-brain barrier, and appears to distribute into tumor tissue. It also demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide and in an MX-1 breast cancer xenograft model both as a single agent and in combination with carboplatin.

  1. Molecular Insights into Poly(ADP-ribose) Recognition and Processing

    PubMed Central

    Žaja, Roko; Mikoč, Andreja; Barkauskaite, Eva; Ahel, Ivan

    2012-01-01

    Poly(ADP-ribosyl)ation is a post-translational protein modification involved in the regulation of important cellular functions including DNA repair, transcription, mitosis and apoptosis. The amount of poly(ADP-ribosyl)ation (PAR) in cells reflects the balance of synthesis, mediated by the PARP protein family, and degradation, which is catalyzed by a glycohydrolase, PARG. Many of the proteins mediating PAR metabolism possess specialised high affinity PAR-binding modules that allow the efficient sensing or processing of the PAR signal. The identification of four such PAR-binding modules and the characterization of a number of proteins utilising these elements during the last decade has provided important insights into how PAR regulates different cellular activities. The macrodomain represents a unique PAR-binding module which is, in some instances, known to possess enzymatic activity on ADP-ribose derivatives (in addition to PAR-binding). The most recently discovered example for this is the PARG protein, and several available PARG structures have provided an understanding into how the PARG macrodomain evolved into a major enzyme that maintains PAR homeostasis in living cells. PMID:24970154

  2. Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress

    PubMed Central

    Gagné, Jean-Philippe; Pic, Émilie; Isabelle, Maxim; Krietsch, Jana; Éthier, Chantal; Paquet, Éric; Kelly, Isabelle; Boutin, Michel; Moon, Kyung-Mee; Foster, Leonard J.; Poirier, Guy G.

    2012-01-01

    Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates. PMID:22669911

  3. Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.

    PubMed

    Gagné, Jean-Philippe; Pic, Emilie; Isabelle, Maxim; Krietsch, Jana; Ethier, Chantal; Paquet, Eric; Kelly, Isabelle; Boutin, Michel; Moon, Kyung-Mee; Foster, Leonard J; Poirier, Guy G

    2012-09-01

    Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates. PMID:22669911

  4. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    SciTech Connect

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  5. Poly(ADP-ribose) in the bone: from oxidative stress signal to structural element.

    PubMed

    Hegedűs, Csaba; Robaszkiewicz, Agnieszka; Lakatos, Petra; Szabó, Éva; Virág, László

    2015-05-01

    Contrary to common perception bone is a dynamic organ flexibly adapting to changes in mechanical loading by shifting the delicate balance between bone formation and bone resorption carried out by osteoblasts and osteoclasts, respectively. In the past decades numerous studies demonstrating production of reactive oxygen or nitrogen intermediates, effects of different antioxidants, and involvement of prototypical redox control mechanisms (Nrf2-Keap1, Steap4, FoxO, PAMM, caspase-2) have proven the central role of redox regulation in the bone. Poly(ADP-ribosyl)ation (PARylation), a NAD-dependent protein modification carried out by poly(ADP-ribose) polymerase (PARP) enzymes recently emerged as a new regulatory mechanism fine-tuning osteoblast differentiation and mineralization. Interestingly PARylation does not simply serve as a signaling mechanism during osteoblast differentiation but also couples it to osteoblast death. Even more strikingly, the poly(ADP-ribose) polymer likely released from succumbed cells at the terminal stage of differentiation is incorporated into the bone matrix representing the first structural role of this versatile biopolymer. Moreover, this new paradigm explains why and how osteodifferentiation and death of cells entering this pathway are closely coupled to each other. Here we review the role of reactive oxygen and nitrogen intermediates as well as PARylation in osteoblast and osteoclast differentiation, function, and cell death. PMID:25660995

  6. Analysis of poly(ADP-ribose) glycohydrolase activity in nuclear extracts from mammalian cells.

    PubMed

    Bernardi, R; Rossi, L; Poirier, G G; Scovassi, A I

    1997-03-01

    We have analysed poly(ADP-ribose) glycohydrolase, the enzyme responsible for in vivo degradation of ADP-ribose polymers, by means of a biochemical assay based on the capacity of the enzyme to use a synthetic 32P-labelled polymer as a substrate. The visualization of the reaction has been achieved by separation of poly and mono(ADP-ribose) by thin-layer chromatography followed by autoradiography, whereas polymer hydrolysis has been quantified by counting the spots corresponding to poly and mono(ADP-ribose). By addition of the enzyme inhibitor ethacridine to the reaction mixture, we have confirmed the specificity of the procedure we have developed. The protocol has been applied to study the specific activity of glycohydrolase in nuclear extracts from different mammalian cell lines and to an apoptotic experimental system, namely HL60 cells treated with etoposide. We have observed the activation of the enzyme after a two-hour drug treatment, that is concomitant with the activation of poly(ADP-ribose) polymerase, the enzyme which synthesizes the polymer. These data suggest a precise regulation of ADP-ribosylation process during cell death by apoptosis. PMID:9074616

  7. Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-ribose)polymerase inhibitors.

    PubMed

    Gangloff, Anthony R; Brown, Jason; de Jong, Ron; Dougan, Douglas R; Grimshaw, Charles E; Hixon, Mark; Jennings, Andy; Kamran, Ruhi; Kiryanov, Andre; O'Connell, Shawn; Taylor, Ewan; Vu, Phong

    2013-08-15

    Structure based drug design of a series of novel 1,4-benzoxazin-3-one derived PARP-1 inhibitors are described. The synthesis, enzymatic & cellular activities and pharmacodynamic effects are described. Optimized analogs demonstrated inhibition of poly-ADP-ribosylation in SW620 tumor bearing nude mice through 24h following a single dose. PMID:23850199

  8. [From poly(ADP-ribose) discovery to PARP inhibitors in cancer therapy].

    PubMed

    Schreiber, Valérie; Illuzzi, Giuditta; Héberlé, Eléa; Dantzer, Françoise

    2015-10-01

    Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by poly(ADP-ribose) polymerases. PARP-1 is a molecular sensor of DNA breaks, playing a key role in the spatial and temporal organization of their repair, contributing to the maintenance of genome integrity and cell survival. The fact that PARP inhibition impairs efficacy of break repair has been exploited as anticancer strategies to potentiate the cytotoxicity of anticancer drugs and radiotherapy. Numerous clinical trials based on this innovative approach are in progress. PARP inhibition has also proved to be exquisitely efficient to kill tumour cells deficient in double strand break repair by homologous recombination, such as cells mutated for the breast cancer early onset genes BRCA1 or BRCA2, by synthetic lethality. Several phase III clinical trials are in progress for the treatment of breast and ovarian cancers with BRCA mutations and the PARP inhibitor olaparib has just been approved for advanced ovarian cancers with germline BRCA mutation. This review recapitulates the history from the discovery of poly(ADP-ribosyl)ation reaction to the promising therapeutic applications of its inhibition in innovating anticancer strategies. Benefits, hopes and obstacles are discussed. PMID:26384693

  9. Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

    PubMed

    Halldórsson, Haraldur; Thors, Brynhildur; Thorgeirsson, Gudmundur

    2015-01-01

    To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD. PMID:25774718

  10. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

    PubMed Central

    James, Dominic I.; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A.; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D.; Ogilvie, Donald J.

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

  11. An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

    PubMed

    James, Dominic I; Durant, Stephen; Eckersley, Kay; Fairweather, Emma; Griffiths, Louise A; Hamilton, Nicola; Kelly, Paul; O'Connor, Mark; Shea, Kerry; Waddell, Ian D; Ogilvie, Donald J

    2016-01-01

    After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years. PMID:27610220

  12. Poly (ADP-ribose) in the pathogenesis of Parkinson's disease

    PubMed Central

    Lee, Yunjong; Kang, Ho Chul; Lee, Byoung Dae; Lee, Yun-Il; Kim, Young Pil; Shin, Joo-Ho

    2014-01-01

    The defining feature of Parkinson’s disease is a progressive and selective demise of dopaminergic neurons. A recent report on Parkinson’s disease animal model demonstrates that poly (ADP-ribose) (PAR) dependent cell death, also named parthanatos, is accountable for selective dopaminergic neuronal loss. Parthanatos is a programmed necrotic cell death, characterized by PARP1 activation, apoptosis inducing factor (AIF) nuclear translocation, and large scale DNA fragmentation. Besides cell death regulation via interaction with AIF, PAR molecule mediates diverse cellular processes including genomic stability, cell division, transcription, epigenetic regulation, and stress granule formation. In this review, we will discuss the roles of PARP1 activation and PAR molecules in the pathological processes of Parkinson’s disease. Potential interaction between PAR molecule and Parkinson’s disease protein interactome are briefly introduced. Finally, we suggest promising points of therapeutic intervention in the pathological PAR signaling cascade to halt progression in Parkinson’s disease. [BMB Reports 2014; 47(8): 424-432] PMID:24874851

  13. Profiling of Biomarkers for the Exposure of Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3, Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy Number Are Identified as Universal Biomarkers

    PubMed Central

    Kim, Hwan-Young; Kim, Hye-Ran; Trang, Nguyen Thi Dai; Baek, Hee-Jo; Moon, Jae-Dong; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook; Kook, Hoon; Shin, Myung-Geun

    2014-01-01

    This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs. PMID:25114913

  14. The Guanine-Quadruplex Structure in the Human c-myc Gene's Promoter Is Converted into B-DNA Form by the Human Poly(ADP-Ribose)Polymerase-1

    PubMed Central

    Fekete, Anna; Kenesi, Erzsebet; Hunyadi-Gulyas, Eva; Durgo, Hajnalka; Berko, Barbara; Dunai, Zsuzsanna A.; Bauer, Pal I.

    2012-01-01

    The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III1 region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes. PMID:22880082

  15. Txk, a member of the non-receptor tyrosine kinase of the Tec family, forms a complex with poly(ADP-ribose) polymerase 1 and elongation factor 1α and regulates interferon-γ gene transcription in Th1 cells

    PubMed Central

    Maruyama, T; Nara, K; Yoshikawa, H; Suzuki, N

    2007-01-01

    We have found previously that Txk, a member of the Tec family tyrosine kinases, is involved importantly in T helper 1 (Th1) cytokine production. However, how Txk regulates interferon (IFN)-γ gene transcription in human T lymphocytes was not fully elucidated. In this study, we identified poly(ADP-ribose) polymerase 1 (PARP1) and elongation factor 1α (EF-1α) as Txk-associated molecules that bound to the Txk responsive element of the IFN-γ gene promoter. Txk phosphorylated EF-1α and PARP1 formed a complex with them, and bound to the IFN-γ gene promoter in vitro. In particular, the N terminal region containing the DNA binding domain of PARP1 was important for the trimolecular complex formation involving Txk, EF-1α and PARP1. Several mutant Txk which lacked kinase activity were unable to form the trimolecular complex. A PARP1 inhibitor, PJ34, suppressed IFN-γ but not interleukin (IL)-4 production by normal peripheral blood lymphocytes (PBL). Multi-colour confocal analysis revealed that Txk and EF-1α located in the cytoplasm in the resting condition. Upon activation, a complex involving Txk, EF-1α and PARP1 was formed and was located in the nucleus. Collectively, Txk in combination with EF-1α and PARP1 bound to the IFN-γ gene promoter, and exerted transcriptional activity on the IFN-γ gene. PMID:17177976

  16. Tetrandrine induces apoptosis Via caspase-8, -9, and -3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin-1/ LC3-I, II signaling pathways in human oral cancer HSC-3 cells.

    PubMed

    Yu, Fu-Shun; Yu, Chun-Shu; Chen, Jaw-Chyun; Yang, Jiun-Long; Lu, Hsu-Feng; Chang, Shu-Jen; Lin, Meng-Wei; Chung, Jing-Gung

    2016-04-01

    Tetrandrine is a bisbenzylisoquinoline alkaloid that was found in the Radix Stephania tetrandra S Moore. It had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC-3 cells in vitro. Treatments of HSC-3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration-dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases-3, -8, and -9, and cleavage of poly (ADP ribose) polymerase (PARP) that were associated with apoptosis, and it also enhanced the expression of LC3-I and -II that were associated with the induction of autophagy in human squamous carcinoma cell line (HSC-3) cells. Tetrandrine induced autophagy in HSC-3 cells was significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre-treatment that confirmed tetrandrine induced cell death may be associated with the autophagy. In conclusion, we suggest that tetrandrine induced cell death may be through the induction of apoptosis as well as autophagy in human oral cancer HSC-3 cells via PARP, caspases/Becline I/LC3-I/II signaling pathways. PMID:25266202

  17. Discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and -2 mutant tumors.

    PubMed

    Jones, Philip; Altamura, Sergio; Boueres, Julia; Ferrigno, Federica; Fonsi, Massimiliano; Giomini, Claudia; Lamartina, Stefania; Monteagudo, Edith; Ontoria, Jesus M; Orsale, Maria Vittoria; Palumbi, Maria Cecilia; Pesci, Silvia; Roscilli, Giuseppe; Scarpelli, Rita; Schultz-Fademrecht, Carsten; Toniatti, Carlo; Rowley, Michael

    2009-11-26

    We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer. PMID:19873981

  18. Discovery and Structure–Activity Relationship of Novel 2,3-Dihydrobenzofuran-7-carboxamide and 2,3-Dihydrobenzofuran-3(2H)-one-7-carboxamide Derivatives as Poly(ADP-ribose)polymerase-1 Inhibitors

    PubMed Central

    2015-01-01

    Novel substituted 2,3-dihydrobenzofuran-7-carboxamide (DHBF-7-carboxamide) and 2,3-dihydrobenzofuran-3(2H)-one-7-carboxamide (DHBF-3-one-7-carboxamide) derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy resulted in lead compound 3 (DHBF-7-carboxamide; IC50 = 9.45 μM). To facilitate synthetically feasible derivatives, an alternative core was designed, DHBF-3-one-7-carboxamide (36, IC50 = 16.2 μM). The electrophilic 2-position of this scaffold was accessible for extended modifications. Substituted benzylidene derivatives at the 2-position were found to be the most potent, with 3′,4′-dihydroxybenzylidene 58 (IC50 = 0.531 μM) showing a 30-fold improvement in potency. Various heterocycles attached at the 4′-hydroxyl/4′-amino of the benzylidene moiety resulted in significant improvement in inhibition of PARP-1 activity (e.g., compounds 66–68, 70, 72, and 73; IC50 values from 0.718 to 0.079 μM). Compound 66 showed selective cytotoxicity in BRCA2-deficient DT40 cells. Crystal structures of three inhibitors (compounds (−)-13c, 59, and 65) bound to a multidomain PARP-1 structure were obtained, providing insights into further development of these inhibitors. PMID:24922587

  19. Active site fingerprinting and pharmacophore screening strategies for the identification of dual inhibitors of protein kinase C [Formula: see text] and poly (ADP-ribose) polymerase-1 (PARP-1).

    PubMed

    Chadha, Navriti; Silakari, Om

    2016-08-01

    Current clinical studies have revealed that diabetic complications are multifactorial disorders that target two or more pathways. The majority of drugs in clinical trial target aldose reductase and protein kinase C ([Formula: see text]), while recent studies disclosed a significant role played by poly (ADP-ribose) polymerase-1 (PARP-1). In light of this, the current study was aimed to identify novel dual inhibitors of [Formula: see text] and PARP-1 using a pharmaco-informatics methodology. Pharmacophore-based 3D QSAR models for these two targets were generated using HypoGen and used to screen three commercially available chemical databases to identify dual inhibitors of [Formula: see text] and PARP-1. Overall, 18 hits were obtained from the screening process; the hits were filtered based on their drug-like properties and predicted binding affinities (docking analysis). Important amino acid residues were predicted by developing a fingerprint of the active site using alanine-scanning mutagenesis and molecular dynamics. The stability of the complexes (18 hits with both proteins) and their final binding orientations were investigated using molecular dynamics simulations. Thus, novel hits have been predicted to have good binding affinities for [Formula: see text] and PARP-1 proteins, which could be further investigated for in vitro/in vivo activity. PMID:27216445

  20. Nitric oxide and superoxide anion differentially activate poly(ADP-ribose) polymerase-1 and Bax to induce nuclear translocation of apoptosis-inducing factor and mitochondrial release of cytochrome c after spinal cord injury.

    PubMed

    Wu, Kay L H; Hsu, Chin; Chan, Julie Y H

    2009-07-01

    We reported previously that complete spinal cord transection (SCT) results in depression of mitochondrial respiratory chain enzyme activity that triggers apoptosis via sequential activations of apoptosis-inducing factor (AIF)- and caspase-dependent cascades in the injured spinal cord. This study tested the hypothesis that nitric oxide (NO) and superoxide anion (O(2)(.-)) serve as the interposing signals between SCT and impaired mitochondrial respiratory functions. Adult Sprague-Dawley rats manifested a significant increase in NO or O(2)(.-) level in the injured spinal cord during the first 3 days after SCT. The augmented O(2)(.-) production, along with concomitant reduction in mitochondrial respiratory chain enzyme activity or ATP level, nuclear translocation of AIF, cytosolic release of cytochrome c, and DNA fragmentation were reversed by osmotic minipump infusion of a NO trapping agent, carboxy-PTIO, or a superoxide dismutase mimetic, tempol, into the epicenter of the transected spinal cord. Intriguingly, carboxy-PTIO significantly suppressed upregulation of poly(ADP-ribose) polymerase-1 (PARP-1) in the nucleus, attenuated nuclear translocation of AIF, inhibited mitochondrial translocation of Bax and antagonized mitochondrial release of cytochrome c; whereas tempol only inhibited the later two cellular events after SCT. We conclude that overproduction of NO and O(2)(.-) in the injured spinal cord promulgates mitochondrial dysfunction and triggers AIF- and caspase-dependent apoptotic signaling cascades via differential upregulation of nuclear PARP-1 and mitochondrial translocation of Bax. PMID:19473058

  1. Targeting poly(ADP-ribose) polymerase and the c-Myb-regulated DNA damage response pathway in castration-resistant prostate cancer.

    PubMed

    Li, Likun; Chang, Wenjun; Yang, Guang; Ren, Chengzhen; Park, Sanghee; Karantanos, Theodoros; Karanika, Styliani; Wang, Jianxiang; Yin, Jianhua; Shah, Parantu K; Takahiro, Hirayama; Dobashi, Masato; Zhang, Wenling; Efstathiou, Eleni; Maity, Sankar N; Aparicio, Ana M; Li Ning Tapia, Elsa M; Troncoso, Patricia; Broom, Bradley; Xiao, Lianchun; Lee, Hyun-Sung; Lee, Ju-Seog; Corn, Paul G; Navone, Nora; Thompson, Timothy C

    2014-05-20

    Androgen deprivation is the standard treatment for advanced prostate cancer (PCa), but most patients ultimately develop resistance and tumor recurrence. We found that MYB is transcriptionally activated by androgen deprivation therapy or genetic silencing of the androgen receptor (AR). MYB silencing inhibited PCa growth in culture and xenografts in mice. Microarray data revealed that c-Myb and AR shared a subset of target genes that encode DNA damage response (DDR) proteins, suggesting that c-Myb may supplant AR as the dominant regulator of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures including AR, MYB, and their common DDR-associated target genes positively correlated with metastasis, castration resistance, tumor recurrence, and decreased survival in PCa patients. In culture and in xenograft-bearing mice, a combination strategy involving the knockdown of MYB, BRCA1, or TOPBP1 or the abrogation of cell cycle checkpoint arrest with AZD7762, an inhibitor of the checkpoint kinase Chk1, increased the cytotoxicity of the poly[adenosine 5'-diphosphate (ADP)-ribose] polymerase (PARP) inhibitor olaparib in PCa cells. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the DDR pathway involving c-Myb, TopBP1, ataxia telangiectasia mutated- and Rad3-related (ATR), and Chk1. PMID:24847116

  2. Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways.

    PubMed

    Ghosh, Rajib; Roy, Sanchita; Kamyab, Johan; Dantzer, Francoise; Franco, Sonia

    2016-09-01

    In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells. PMID:27373144

  3. Poly(ADP-ribose) metabolism in young and old cells: response to cellular stresses

    SciTech Connect

    Gracy, R.W.; Sims, J.L.; Cini, J.

    1986-05-01

    The authors have examined the effect of several cellular stresses on poly(ADP-ribose) metabolism in human fibroblasts of low passage number derived from young and old donors. Poly(ADP-ribose) was synthesized in response to alkylation of DNA caused by N-methyl-N'-nitro-N-nitroguanidine and by hyperthermic treatment at 43 /sup 0/C or 45 /sup 0/C. Ethanol is able to potentiate poly(ADP-ribose) accumulation following these treatments. There was little if any difference in the response of young and old cells to these stresses. Amino acid analogs are thought to induce a response in mammalian cells similar to that caused by hyperthermia and ethanol. However, amino acid analogs such as L-azetidine did not produce effects on poly(ADP-ribose) metabolism like those produced by ethanol or hyperthermia. The authors have also examined the poly(ADP-ribose) content of bovine eye lens. Each eye lens contains populations of young and old cells that are distributed in a fixed graduated manner. Thus, the eye lens allows the study of aging cells with identical genetic backgrounds without the complications of serial passage of young cultures in in vitro aging systems.

  4. Affinity-Based Assays for the Identification and Quantitative Evaluation of Noncovalent Poly(ADP-Ribose)-Binding Proteins

    PubMed Central

    Gagné, Jean-Philippe; Haince, Jean-François; Pic, Émilie; Poirier, Guy G.

    2014-01-01

    Poly(ADP-ribose) polymerases have been linked to several cellular functions, most of which being mediated through the dynamics of poly(ADP-ribose) (pADPr). In several pathways, pADPr is the effector molecule that regulates cellular signaling and dictates biological outcomes. pAPDr is a central molecule that is capable of promoting both cell survival through the maintenance of genome integrity and cell death that occurs by way of a signal-mediated apoptotic-like process. Thus, interactions with pADPr are extremely important in bringing about the balanced regulation that controls cell fate. Further clues regarding these functions are emerging from a growing list of proteins with which pADPr interacts. Here, we describe the current approaches for investigating noncovalent protein interactions with pADPr. PMID:21870257

  5. Discovery and structure-activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADP-ribose) glycohydrolase (PARG)

    PubMed Central

    Steffen, Jamin D.; Coyle, Donna L.; Damodaran, Komath; Beroza, Paul; Jacobson, Myron K.

    2011-01-01

    The metabolism of poly(ADP-ribose) (PAR) in response to DNA strand breaks, which involves the concerted activities of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG), modulates cell recovery or cell death depending upon the level of DNA damage. While PARP inhibitors show high promise in clinical trials due to their low toxicity and selectivity for BRCA related cancers, evaluation of the therapeutic potential of PARG is limited by the lack of well-validated cell permeable inhibitors. In this study, Target-related Affinity Profiling (TRAP), an alternative to high-throughput screening, was used to identify a number of drug-like compounds from several chemical classes that demonstrated PARG inhibition in the low-micromolar range. A number of analogs of one of the most active chemotypes were synthesized to explore structure-activity relationship (SAR) for that series. This led to the discovery of a putative pharmacophore for PARG inhibition that contains a modified salicylanilide structure. Interestingly, these compounds also inhibit PARP-1, indicating strong homology in the active sites of PARG and PARP-1, and raising a new challenge for development of PARG specific inhibitors. The cellular activity of a lead inhibitor was demonstrated by the inhibition of both PARP and PARG activity in squamous cell carcinoma cells, although preferential inhibition of PARG relative to PARP was observed. The ability of inhibitors to modulate PAR metabolism via simultaneous effects on PARPs and PARG may represent a new approach for therapeutic development. PMID:21692479

  6. Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

    NASA Astrophysics Data System (ADS)

    Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

    2013-12-01

    Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

  7. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

    PubMed Central

    Luo, Xin; Kraus, W. Lee

    2012-01-01

    Cellular stress responses are mediated through a series of regulatory processes that occur at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These responses require a complex network of sensors and effectors from multiple signaling pathways, including the abundant and ubiquitous nuclear enzyme poly(ADP-ribose) (PAR) polymerase-1 (PARP-1). PARP-1 functions at the center of cellular stress responses, where it processes diverse signals and, in response, directs cells to specific fates (e.g., DNA repair vs. cell death) based on the type and strength of the stress stimulus. Many of PARP-1's functions in stress response pathways are mediated by its regulated synthesis of PAR, a negatively charged polymer, using NAD+ as a donor of ADP-ribose units. Thus, PARP-1's functions are intimately tied to nuclear NAD+ metabolism and the broader metabolic profile of the cell. Recent studies in cell and animal models have highlighted the roles of PARP-1 and PAR in the response to a wide variety of extrinsic and intrinsic stress signals, including those initiated by oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, and metabolic stresses. These responses underlie pathological conditions, including cancer, inflammation-related diseases, and metabolic dysregulation. The development of PARP inhibitors is being pursued as a therapeutic approach to these conditions. In this review, we highlight the newest findings about PARP-1's role in stress responses in the context of the historical data. PMID:22391446

  8. Phase 2 multicentre trial investigating intermittent and continuous dosing schedules of the poly(ADP-ribose) polymerase inhibitor rucaparib in germline BRCA mutation carriers with advanced ovarian and breast cancer

    PubMed Central

    Drew, Yvette; Ledermann, Jonathan; Hall, Geoff; Rea, Daniel; Glasspool, Ros; Highley, Martin; Jayson, Gordon; Sludden, Julieann; Murray, James; Jamieson, David; Halford, Sarah; Acton, Gary; Backholer, Zoe; Mangano, Raffaella; Boddy, Alan; Curtin, Nicola; Plummer, Ruth

    2016-01-01

    Background: Rucaparib is an orally available potent selective small-molecule inhibitor of poly(ADP-ribose) polymerase (PARP) 1 and 2. Rucaparib induces synthetic lethality in cancer cells defective in the homologous recombination repair pathway including BRCA-1/2. We investigated the efficacy and safety of single-agent rucaparib in germline (g) BRCA mutation carriers with advanced breast and ovarian cancers. Methods: Phase II, open-label, multicentre trial of rucaparib in proven BRCA-1/2 mutation carriers with advanced breast and or ovarian cancer, WHO PS 0–1 and normal organ function. Intravenous (i.v.) and subsequently oral rucaparib were assessed, using a range of dosing schedules, to determine the safety, tolerability, dose-limiting toxic effects and pharmacodynamic (PD) and pharmacokinetic (PK) profiles. Results: Rucaparib was well tolerated in patients up to doses of 480 mg per day and is a potent inhibitor of PARP, with sustained inhibition ⩾24 h after single doses. The i.v. rucaparib (intermittent dosing schedule) resulted in an objective response rate (ORR) of only 2% but with 41% (18 out of 44) patients achieved stable disease for ⩾12 weeks and 3 patients maintaining disease stabilisation for >52 weeks. The ORR for oral rucaparib (across all six dose levels) was 15%. In the oral cohorts, 81% (22 out of 27) of the patients had ovarian cancer and 12 out of 13, who were dosed continuously, achieved RECIST complete response/partial response (CR/PR) or stable disease (SD) ⩾12 weeks, with a median duration of response of 179 days (range 84–567 days). Conclusions: Rucaparib is well tolerated and results in high levels of PARP inhibition in surrogate tissues even at the lowest dose levels. Rucaparib is active in gBRCA-mutant ovarian cancer and this activity correlates with platinum-free interval. The key lessons learned from this study is that continuous rucaparib dosing is required for optimal response, the recommended phase 2 dose (RP2D) for

  9. Drug repurposing screen identifies lestaurtinib amplifies the ability of the poly (ADP-ribose) polymerase 1 inhibitor AG14361 to kill breast cancer associated gene-1 mutant and wild type breast cancer cells

    PubMed Central

    2014-01-01

    Introduction Breast cancer is a devastating disease that results in approximately 40,000 deaths each year in the USA. Current drug screening and chemopreventatitive methods are suboptimal, due in part to the poor specificity of compounds for cancer cells. Poly (ADP-ribose) polymerase 1 (PARP1) inhibitor (PARPi)-mediated therapy is a promising approach for familial breast cancers caused by mutations of breast cancer-associated gene-1 and -2 (BRCA1/2), yet drug resistance frequently occurs during the treatment. Moreover, PARPis exhibit very little effect on cancers that are proficient for DNA repair and clinical efficacy for PARPis as single-agent therapies has yet to be illustrated. Methods Using a quantitative high-throughput screening approach, we screened a library containing 2,816 drugs, most of which are approved for human or animal use by the Food and Drug Administration (FDA) or other countries, to identify compounds that sensitize breast cancer cells to PARPi. After initial screening, we performed further cellular and molecular analysis on lestaurtinib, which is an orally bioavailable multikinase inhibitor and has been used in clinical trials for myeloproliferative disorders and acute myelogenous leukemia. Results Our study indicated that lestaurtinib is highly potent against breast cancers as a mono-treatment agent. It also strongly enhanced the activity of the potent PARPi AG14361 on breast cancer cell growth both in vitro and in vivo conditions. The inhibition of cancer growth is measured by increased apoptosis and reduced cell proliferation. Consistent with this, the treatment results in activation of caspase 3/7, and accumulation of cells in the G2 phase of the cell cycle, irrespective of their BRCA1 status. Finally, we demonstrated that AG14361 inhibits NF-κB signaling, which is further enhanced by lestaurtinib treatment. Conclusions Lestaurtinib amplifies the ability of the PARP1 inhibitor AG14361 to kill BRCA1 mutant and wild-type breast cancer

  10. PJ34, a poly(ADP-ribose) polymerase (PARP) inhibitor, reverses melphalan-resistance and inhibits repair of DNA double-strand breaks by targeting the FA/BRCA pathway in multidrug resistant multiple myeloma cell line RPMI8226/R.

    PubMed

    Xiong, Ting; Wei, Heng; Chen, Xiaoqiong; Xiao, Hui

    2015-01-01

    There is still no ideal treatment for multidrug resistant multiple myeloma, looking for drugs which can reverse chemotherapy resistance and enhance curative effects of chemotherapy drugs becomes a problem that needs to be solved urgently. Poly(ADP-ribose) polymerase inhibitors appear to be an important tool for medical therapy of several malignancies. In the present study, we investigated the potential of the PARP-1 inhibitor PJ34, in vitro, to further enhance the efficacy of the traditional chemotherapy drug melphalan in the multidrug-resistant multiple myeloma cell line RPMI8226/R. The effects of different concentrations of PJ34 and melphalan on cell proliferation were determined by the CCK-8 assay. The expressions of FA/BRCA pathway-related factors were detected by western blotting and RT-PCR. The percentage of cell apoptosis was measured with flow cytometry. DNA double-strand break (DSB) repair was quantified by γH2AX immunofluorescence. In addition, DNA damage repair at the level of the individual cell was determined by comet assay. Co-administration of PJ34 and melphalan had synergistic inhibitory effects on the proliferation of RPMI8226/R cells, suggesting more powerful antitumor activities. The apoptosis percentage also was increased more obviously by the treatment of melphalan plus PJ34. The activation of FA/BRCA pathway was inhibited by downregulation of related factors including FANCD2, BRCA2 and Rad51. PJ34 significantly increased the ratio of γH2AX-positive cells and the number of foci/cells. The comet tail rate of cells, tail length, tail moment and Olive tail moment all increased after PJ34 treatment in RPMI8226/R cells. These results indicate that PJ34 combined treatment with melphalan produces synergistic effects and reverses multidrug resistance of RPMI8226/R cells effectively. PJ34 cannot induce DNA damage directly, but it may increase the DNA damage induced by melphalan through inhibiting DNA damage repair. The suppression of FA

  11. Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

    PubMed Central

    Barkauskaite, Eva; Brassington, Amy; Tan, Edwin S.; Warwicker, Jim; Dunstan, Mark S.; Banos, Benito; Lafite, Pierre; Ahel, Marijan; Mitchison, Timothy J.; Ahel, Ivan; Leys, David

    2013-01-01

    Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios. PMID:23917065

  12. Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

    PubMed Central

    Vilchez Larrea, Salomé C.; Schlesinger, Mariana; Kevorkian, María L.; Flawiá, Mirtha M.; Alonso, Guillermo D.; Fernández Villamil, Silvia H.

    2013-01-01

    Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease. PMID:23776710

  13. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function

    PubMed Central

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L.; Hanzlikova, Hana; Oliver, Antony W.; Caldecott, Keith W.

    2015-01-01

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  14. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

    PubMed

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L; Hanzlikova, Hana; Oliver, Antony W; Caldecott, Keith W

    2015-08-18

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  15. Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation

    PubMed Central

    Huang, Peiwu; Zhuang, Zhixiong; Liu, Jianjun; Gao, Wei; Liu, Yinpin; Huang, Haiyan

    2016-01-01

    Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer. PMID:27003318

  16. Poly(ADP-Ribose) Glycohydrolase (PARG) Silencing Suppresses Benzo(a)pyrene Induced Cell Transformation.

    PubMed

    Li, Xuan; Li, Xiyi; Zhu, Zhiliang; Huang, Peiwu; Zhuang, Zhixiong; Liu, Jianjun; Gao, Wei; Liu, Yinpin; Huang, Haiyan

    2016-01-01

    Benzo(a)pyrene (BaP) is a ubiquitously distributed environmental pollutant and known carcinogen, which can induce malignant transformation in rodent and human cells. Poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme that catalyzes the degradation of poly(ADP-ribose) (PAR), has been known to play an important role in regulating DNA damage repair and maintaining genomic stability. Although PARG has been shown to be a downstream effector of BaP, the role of PARG in BaP induced carcinogenesis remains unclear. In this study, we used the PARG-deficient human bronchial epithelial cell line (shPARG) as a model to examine how PARG contributed to the carcinogenesis induced by chronic BaP exposure under various concentrations (0, 10, 20 and 40 μM). Our results showed that PARG silencing dramatically reduced DNA damages, chromosome abnormalities, and micronuclei formations in the PARG-deficient human bronchial epithelial cells compared to the control cells (16HBE cells). Meanwhile, the wound healing assay showed that PARG silencing significantly inhibited BaP-induced cell migration. Furthermore, silencing of PARG significantly reduced the volume and weight of tumors in Balb/c nude mice injected with BaP induced transformed human bronchial epithelial cells. This was the first study that reported evidences to support an oncogenic role of PARG in BaP induced carcinogenesis, which provided a new perspective for our understanding in BaP exposure induced cancer. PMID:27003318

  17. Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

    SciTech Connect

    Meyer, Ralph G. . E-mail: meyerg@vet.upenn.edu; Meyer-Ficca, Mirella L.; Whatcott, Clifford J.; Jacobson, Elaine L.; Jacobson, Myron K.

    2007-08-01

    Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of {approx} 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.

  18. Dual inhibitory effects of dimethyl sulfoxide on poly(ADP-ribose) synthetase.

    PubMed

    Banasik, M; Ueda, K

    1999-01-01

    Dimethyl sulfoxide (DMSO), a solvent popularly used for dissolving water-insoluble compounds, is a weak inhibitor of poly(ADP-ribose) synthetase, that is a nuclear enzyme producing (ADP-ribose)n from NAD+. The inhibitory mode and potency depend on the concentration of substrate, NAD+, as well as the temperature of the reaction; at micromolar concentrations of NAD+, the inhibition by DMSO is biphasic at 37 degrees C, but is monophasic and apparently competitive with NAD+ at 25 degrees C. DMSO, on the other hand, diminishes dose-dependently and markedly the inhibitory potency of benzamide and other inhibitors. Other organic solvents, ethanol and methanol, also show a biphasic effect on the synthetase activity at different concentrations. PMID:10445046

  19. A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.

    PubMed

    Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, Neil; Truman, Caroline; Hardern, Ian M; Ogilvie, Donald J

    2016-06-15

    Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition. PMID:27036617

  20. Readers of poly(ADP-ribose): designed to be fit for purpose

    PubMed Central

    Teloni, Federico; Altmeyer, Matthias

    2016-01-01

    Post-translational modifications (PTMs) regulate many aspects of protein function and are indispensable for the spatio-temporal regulation of cellular processes. The proteome-wide identification of PTM targets has made significant progress in recent years, as has the characterization of their writers, readers, modifiers and erasers. One of the most elusive PTMs is poly(ADP-ribosyl)ation (PARylation), a nucleic acid-like PTM involved in chromatin dynamics, genome stability maintenance, transcription, cell metabolism and development. In this article, we provide an overview on our current understanding of the writers of this modification and their targets, as well as the enzymes that degrade and thereby modify and erase poly(ADP-ribose) (PAR). Since many cellular functions of PARylation are exerted through dynamic interactions of PAR-binding proteins with PAR, we discuss the readers of this modification and provide a synthesis of recent findings, which suggest that multiple structurally highly diverse reader modules, ranging from completely folded PAR-binding domains to intrinsically disordered sequence stretches, evolved as PAR effectors to carry out specific cellular functions. PMID:26673700

  1. Bioorthogonally Functionalized NAD(+) Analogues for In-Cell Visualization of Poly(ADP-Ribose) Formation.

    PubMed

    Wallrodt, Sarah; Buntz, Annette; Wang, Yan; Zumbusch, Andreas; Marx, Andreas

    2016-06-27

    Poly(ADP-ribos)ylation (PARylation) is a major posttranslational modification and signaling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and its binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD(+) onto various acceptor proteins. Molecular studies of PARylation thus remain challenging. Herein, we present the development of bioorthogonally functionalized NAD(+) analogues for the imaging of PARylation in vitro and in cells. Our results show that 2-modified NAD(+) analogues perform remarkably well and can be applied to the in-cell visualization of PARylation simultaneously in two colors. This tool gives insight into the substrate scope of ARTs and will help to further elucidate the biological role of PARylation by offering fast optical, multichannel read-outs. PMID:27080423

  2. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

    PubMed Central

    Altmeyer, Matthias; Neelsen, Kai J.; Teloni, Federico; Pozdnyakova, Irina; Pellegrino, Stefania; Grøfte, Merete; Rask, Maj-Britt Druedahl; Streicher, Werner; Jungmichel, Stephanie; Nielsen, Michael Lund; Lukas, Jiri

    2015-01-01

    Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation. PMID:26286827

  3. Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders.

    PubMed

    Horkay, I; Topaloglou, A; Teherani, D K; Kósa, A; Altmann, H

    1990-01-01

    Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses. PMID:2094134

  4. Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

    PubMed Central

    Tucker, Julie A.; Bennett, Neil; Brassington, Claire; Durant, Stephen T.; Hassall, Giles; Holdgate, Geoff; McAlister, Mark; Nissink, J. Willem M.; Truman, Caroline; Watson, Martin

    2012-01-01

    Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors. PMID:23251397

  5. A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

    SciTech Connect

    Whatcott, Clifford J.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Myron K.

    2009-12-10

    Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.

  6. Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

    PubMed Central

    Pellegrino, Stefania; Altmeyer, Matthias

    2016-01-01

    Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

  7. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

    PubMed

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-12-15

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  8. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response

    PubMed Central

    Zhang, Feng; Shi, Jiazhong; Chen, Shih-Hsun; Bian, Chunjing; Yu, Xiaochun

    2015-01-01

    Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response. PMID:26400172

  9. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

    PubMed Central

    D'Amours, D; Desnoyers, S; D'Silva, I; Poirier, G G

    1999-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism. PMID:10455009

  10. Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death.

    PubMed

    Andrabi, Shaida A; Kang, Ho Chul; Haince, Jean-François; Lee, Yun-Il; Zhang, Jian; Chi, Zhikai; West, Andrew B; Koehler, Raymond C; Poirier, Guy G; Dawson, Ted M; Dawson, Valina L

    2011-06-01

    Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders. PMID:21602803

  11. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    SciTech Connect

    Nakadate, Yusuke; Kodera, Yasuo; Kitamura, Yuka; Tachibana, Taro; Tamura, Tomohide; Koizumi, Fumiaki

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  12. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint.

    PubMed

    Nakadate, Yusuke; Kodera, Yasuo; Kitamura, Yuka; Tachibana, Taro; Tamura, Tomohide; Koizumi, Fumiaki

    2013-11-29

    Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy. PMID:24211580

  13. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    SciTech Connect

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  14. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells.

    PubMed

    Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki; Ida, Chieri; Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke; Moss, Joel; Miwa, Masanao

    2016-08-01

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24-72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  15. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  16. Benadrostin, new inhibitor of poly(ADP-ribose) synthetase, produced by actinomycetes. I. Taxonomy, production, isolation, physico-chemical properties and biological activities.

    PubMed

    Aoyagi, T; Yoshida, S; Harada, S; Okuyama, A; Nakayama, C; Yoshida, T; Hamada, M; Takeuchi, T; Umezawa, H

    1988-08-01

    Benadrostin, a new inhibitor of poly(ADP-ribose) synthetase was discovered in the fermentation broth of Streptomyces flavovirens MH499-O'F1. It was purified by chromatography followed by solvent extraction and then isolated as colorless prisms. Benadrostin has the molecular formula of C8H5NO4. It was competitive with the substrate, and the inhibition constant (Ki) was 34 microM. PMID:3139601

  17. Approche morphologique de la fragmentation de l'ADN radio-induite par immunomarquage anti-poly (ADP-ribose) polymérase (PARP) : étude de cultures d'oligodendrogliomes

    NASA Astrophysics Data System (ADS)

    Varlet, P.; Beuvon, F.; Cervera, P.; Averbeck, D.; Daumas-Duport, C.

    1998-04-01

    Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme encompassing two zinc finger motifs which specifically binds to radiation induced DNA strand breaks. We develop a new immuno-labelling of poly ADP-ribose which coupled together with the immunodetection of cells in cycle with MIB1, permits to detect and quantify the DNA fragmentation induced by radiations (Cesium137). This method, applied to organotypical cultures of human oligodendroglioma, submitted to radiation, a dose dependant nuclear signal. This one increased significantly in the presence of a radiosensitizer like iododeoxyuridine (IUDR 5 g/ml). This poly ADP-ribose immunodetection can be useful, to detect furtherly the individual radiosensitivity of human glioma. Les protéases “ICE-like" ou caspases, sont les homologues humaines du produit du gène ced-3 du ver Caenorhabditis elegans et sont activées lors des étapes précoces de l'apoptose. L'objectif de ce travail vise à déterminer dans quelle mesure l'inhibition de l'une d'entre elles, la caspase-3 est susceptible de modifier la sensibilité des cellules vis-à-vis de l'apoptose radioinduite. Des lymphocytes spléniques murins irradiés en présence de Ac-DVED-CHO un inhibiteur spécifique de la caspase-3 présentent un taux de particules hypodiploïdes radioinduites bien inférieur à celui des contrôles et une diminution drastique de la fragmentation internucléosomale de l'ADN. Toutefois, ni l'externalisation des phospholipides anioniques, autre marqueur spécifique de l'apoptose, ni la viabilité ne sont affectées.

  18. Molecular biology basis for the response of poly(ADP-rib) polymerase and NAD metabolism to dna damage caused by mustard alkylating agents. Final report, 30 April 1990-30 July 1994

    SciTech Connect

    Smulson, M.E.

    1994-08-30

    During the course of this contract, we have performed a variety of experiments whose intent has been to provide a strategy to modulate the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) in cultured keratinocytes. During this study, human keratinocyte lines were stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA by dexamethasone in cultured cells selectively lowered levels of PADPRP in RNA, protein, and enzyme activity. Induction of antisense RNA led to a reduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to an alkylating agent. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a sulfur mustard to the grafted transfected skin layers. Accordingly, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well.

  19. Ring finger protein 146/Iduna is a Poly (ADP-ribose) polymer binding and PARsylation dependent E3 ubiquitin ligase

    PubMed Central

    Zhou, Zhi-dong; Chan, Christine Hui-shan; Xiao, Zhi-cheng

    2011-01-01

    Recent findings suggest that Ring finger protein 146 (RNF146), also called Iduna, have neuroprotective property due to its inhibition of Parthanatos via binding with Poly(ADP-ribose) (PAR). The Parthanatos is a PAR dependent cell death that has been implicated in many human diseases. RNF146/Iduna acts as a PARsylation-directed E3 ubquitin ligase to mediate tankyrase-dependent degradation of axin, thereby positively regulates Wnt signaling. RNF146/Iduna can also facilitate DNA repair and protect against cell death induced by DNA damaging agents or γ-irradiation. It can translocate to the nucleus after cellular injury and promote the ubiquitination and degradation of various nuclear proteins involved in DNA damage repair. The PARsylation-directed ubquitination mediated by RNF146/Iduna is analogous to the phosphorylation-directed ubquitination catalyzed by Skp1-Cul1-F-box (SCF) E3 ubiquitin complex. RNF146/Iduna has been found to be implicated in neurodegenerative disease and cancer development. Therefore modulation of the PAR-binding and PARsylation dependent E3 ligase activity of RNF146/Iduna could have therapeutic significance for diseases, in which PAR and PAR-binding proteins play key pathophysiologic roles. PMID:22274711

  20. Mono-galloyl glucose derivatives are potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors and partially reduce PARP-1-dependent cell death

    PubMed Central

    Formentini, L; Arapistas, P; Pittelli, M; Jacomelli, M; Pitozzi, V; Menichetti, S; Romani, A; Giovannelli, L; Moroni, F; Chiarugi, A

    2008-01-01

    Background and purpose: Maintenance of poly(ADP-ribose) (PAR) polymers at homoeostatic levels by PAR glycohydrolase (PARG) is central in cell functioning and survival. Yet the pharmacological relevance of PARG inhibitors is still debated. Gallotannin, a complex mixture of hydrolysable tannins from oak gall, inhibits PARG but which of its constituents is responsible for the inhibition and whether the pharmacodynamic properties are due to its antioxidant properties, has not yet been established. Experimental approach: A structure–activity relationship study was conducted on different natural and synthetic tannins/galloyl derivatives as potential PARG inhibitors, using a novel in vitro enzymic assay. Cytotoxicity was assayed in cultured HeLa cells. Key results: Mono-galloyl glucose compounds were potent inhibitors of PARG, with activities similar to that of ADP-(hydroxymethyl) pyrrolidinediol, the most potent PARG inhibitor yet identified. When tested on HeLa cells exposed to the PAR polymerase (PARP)-1-activating compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), 3-galloyl glucose weakly inhibited PAR degradation. Conversely, the more lipophilic, 3-galloyl-1,2-O-isopropylidene glucose, despite being inactive on the pure enzyme, efficiently prolonged the half-life of the polymers in intact HeLa cells. Also, PARG inhibitors, but not radical scavengers, reduced, in part, cell death caused by MNNG. Conclusions and implications: Taken together, our findings identify mono-galloyl glucose derivatives as potent PARG inhibitors, and emphasize the active function of this enzyme in cell death. PMID:18806807

  1. Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

    PubMed Central

    Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V. V.; Intorne, Aline C.; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A.; Shan, Libo; He, Ping

    2015-01-01

    Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks. PMID:25569773

  2. Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

    PubMed Central

    Virág, L; Scott, G S; Cuzzocrea, S; Marmer, D; Salzman, A L; Szabó, C

    1998-01-01

    The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis

  3. Molecular biological basis for the response of poly(ADP-rib) polymerase and NAD metabolism to DNA damage caused by mustard alkylating agents. Midterm report

    SciTech Connect

    Smulson, M.E.

    1996-07-01

    During the course of this contract, we have performed a variety of experiments to provide a strategy to modulate the nuclear enzyme poly(ADP-ribose) polymerase (PARP), in cultured keratinocytes. This enzyme modifies a variety of nuclear proteins utilizing NAD. DNA is required for the catalytic activity of the enzyme and the activity is dependent upon the presence of strand breaks in this DNA. It has been hypothesized that human skin exposed to mustards may develop blisters due to a generalized lowering of NAD in exposed skin cells. During the contract period, we have established a stably transfected human keratinocyte cell line which expresses antisense transcripts to PARP mRNA when these keratinocyte were grafted onto nude mice they formed histologically normal human skin. Accordingly, a model system has been developed in which the levels of PARP can be selectively manipulated in human keratinocytes in reconstituted epidermis as well. We also showed that PARP was proteolytically cleaved at the onset of spontaneous apoptosis following proteolytic conversion of CPP32b to its active form, termed `apopain`. Having characterized the events associated with apoptosis, we determined, during the last period, whether any or all of these features could be observed following exposure of keratinocytes to SM.

  4. Studies on protein poly(ADP-ribosylation) using high resolution gel electrophoresis.

    PubMed

    Boulikas, T

    1990-08-25

    Analysis of poly(ADP-ribose) synthesized in cellular lysates or in isolated nuclei on 100-cm-long thin gels of 20% polyacrylamide, 2.5 M urea permits determination of the exact size of poly(ADP-ribose) molecules using labeled oligonucleotides as molecular weight markers. The size and concentration of poly(ADP-ribose) molecules increase at time intervals during its synthesis. Differences in the concentration of poly(ADP-ribose) size classes among cell lines are also shown. Inhibition of poly(ADP-ribose) degradation by ethacridine that directly interacts with the polymer and inhibits its hydrolysis by poly(ADP-ribose) glycohydrolase shows a dramatic increase in both polymer size and concentration. Use of alkaline conditions for the hydrolysis of poly(ADP-ribose)-protein linkages reveals a specific shortening of all size classes of poly(ADP-ribose) compared with its size in preparations obtained by extensive digestion of nuclei with nucleases, RNases, and proteases. PMID:2167322

  5. Fragmentation radioinduite de l'ADN et réparation étudiée par immunomarquage anti poly(ADP-ribose)polymérase (PARP) dans les cellules de hamster chinois (CHO)

    NASA Astrophysics Data System (ADS)

    Bidon, N.; Varlet, P.; Noël, G.; Demurcia, G.; Salamero, J.; Averbeck, D.

    1998-04-01

    The poly(ADP-ribose)polymerase is a nuclear ubiquitous enzyme capable of binding to DNA breaks. Chinese hamster ovary cells were (CHO-K1) cultured on slides and γ-irradiated (137Cs) at a high (12.8 Gy/min) or medium dose rate (5 Gy/min), and immunolabelling against (ADP-ribose) polymers immediatly or three hours after irradiation. Quantification and localisation of γ-ray induced breaks was performed by confocal microscopy. The results show a dose effect relationship, a dose-rate effect and the signal disappearence after 3 hours at 37 °C. The presence of PARP activity appears to reflect γ-rays induced DNA fragmentation. Le poly(ADP-ribose)polymérase est une enzyme nucléaire ubiquitaire capable de se fixer sur les cassures de l'ADN. Sur une lignée sauvage de cellules d'ovaire de hamster chinois (CHO-K1) cultivée sur lame et irradiée aux rayons γ à haut débit de dose (HD) 12,8 Gy/min ou à moyen débit de dose (MD) 5 Gy/min, nous avons réalisé un immunomarquage anti-polymères d'ADP-ribose immédiatement après l'irradiation γ ou après trois heures d'incubation à 37 °C. La quantification et la localisation des lésions radioinduites ont été réalisées par microscopie confocale. Les résultats montrent une relation dose-effet et un effet de débit de dose, ainsi qu'une disparition du signal après 3 heures à 37 °C. La présence de la PARP semble donc bien refléter la fragmentation radioinduite de l'ADN.

  6. Regulation of chromatin structure by poly(ADP-ribosyl)ation

    PubMed Central

    Beneke, Sascha

    2012-01-01

    The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose) has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose), the zinc-finger protein poly(ADP-ribose) polymerase-1 (PARP1), was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor. PMID:22969794

  7. TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP-ribosyl)ation.

    PubMed Central

    Rawling, J M; Alvarez-Gonzalez, R

    1997-01-01

    We have examined the susceptibility of some of the basal eukaryotic transcription factors as covalent targets for poly(ADP-ribosyl)ation. Human recombinant TATA-binding protein, transcription factor (TF)IIB and TFIIF (made up of the 30 and 74 kDa RNA polymerase II-associated proteins RAP30 and RAP74) were incubated with calf thymus poly(ADP-ribose) polymerase and [32P]NAD+ at 37 degrees C. On lithium dodecyl sulphate/PAGE and autoradiography, two bands of radioactivity, coincident with RAP30 and RAP74, were observed. No radioactivity co-migrated with TATA-binding protein or TFIIB. The phenomenon was dependent on the presence of nicked DNA, which is essential for poly(ADP-ribose) polymerase activity. Covalent modification of TFIIF increased with time of incubation, with increasing TFIIF concentration and with increasing NAD+ concentration. High-resolution PAGE confirmed that the radioactive species associated with RAP30 and RAP74 were ADP-ribose polymers. From these observations, we conclude that both TFIIF subunits are highly specific substrates for covalent poly(ADP-ribosyl)ation. PMID:9164864

  8. Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism

    PubMed Central

    Rotin, Lianne E.; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L.; Minden, Mark D.; Slassi, Malik; Schimmer, Aaron D.

    2016-01-01

    Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease. PMID:26624983

  9. Effect of genetic disruption of poly (ADP-ribose) synthetase on delayed production of inflammatory mediators and delayed necrosis during myocardial ischemia-reperfusion injury.

    PubMed

    Yang, Z; Zingarelli, B; Szabó, C

    2000-01-01

    The nuclear enzyme poly (ADP ribose) synthetase (PARS) has been shown to play an important role in the pathogenesis of various forms of ischemia or reperfusion injury and circulatory shock. Recent studies demonstrated that inhibition or genetic inactivation of PARS is beneficial in the early phase of myocardial reperfusion injury. The aim of the present study was to investigate whether inactivation of PARS influences the delayed myocardial necrosis and the production of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha), the anti-inflammatory cytokine interleukin-10 (IL-10), and the free radical nitric oxide in the late stage of myocardial reperfusion injury. The results demonstrate that genetic disruption of PARS provides marked protection against the delayed myocardial ischemia and reperfusion injury. In addition, in the absence of functional PARS, a suppression of TNFalpha, IL-10, and nitric oxide production was found. These findings provide direct evidence that PARS activation participates in the development of delayed cell injury and delayed mediator production in myocardial reperfusion injury. PMID:10638671

  10. Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism.

    PubMed

    Rotin, Lianne E; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L; Minden, Mark D; Slassi, Malik; Schimmer, Aaron D

    2016-01-19

    Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease. PMID:26624983

  11. Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription

    PubMed Central

    Verdone, Loredana; La Fortezza, Marco; Ciccarone, Fabio; Caiafa, Paola; Zampieri, Michele; Caserta, Micaela

    2015-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription. PMID:26636673

  12. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

    PubMed Central

    Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1986-01-01

    To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

  13. Phosphoproteomic Approach to Characterize Protein Mono- and Poly(ADP-ribosyl)ation Sites from Cells

    PubMed Central

    2015-01-01

    Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome. PMID:24920161

  14. The promising role of poly(ADP-ribose) polymerase inhibitors in prostate cancer

    PubMed Central

    Aragon-Ching, Jeanny B

    2016-01-01

    The TOPARP study showed the beneficial utility of olaparib in a heavily pretreated population of metastatic castration-resistant prostate cancer who were biomarker-positive for aberrations in DNA repair gene. A higher response rate to olaparib of 88% (14 out of 16 patients), time to radiographic progression as well as overall survival, was seen in patients who were biomarker-positive compared to those who were not. This study showed a promising way of targeting prostate cancer with an enriched population of patients who harbor these deleterious genes. This paves the way for offering new therapeutic opportunities for men who have otherwise few remaining options. PMID:26908063

  15. Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity

    PubMed Central

    2014-01-01

    Background Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity. Methods We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay. Results In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control. Conclusion Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability. PMID:24528514

  16. Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

    PubMed Central

    Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as

  17. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  18. 50Years of poly(ADP-ribosyl)ation.

    PubMed

    Virág, László

    2013-12-01

    The seminal paper published in 1963 by Chambon, Weil and Mandel reporting a new NAD-dependent protein modification now known as poly(ADP-ribosyl)ation (PARylation) marked the launch of a new era in both protein research and cell biology. In the coming decades, the identity, biochemical characteristics and regulation of enzymes responsible for the synthesis and degradation of protein-bound poly(ADP-ribose) have been discovered and the surprisingly multifarious biological roles of PARylation have not ceased to amaze cell and molecular biologists ever since. The review series on PARylation following this preface is comprised of ten papers written by great experts of the field and aims to provide practicing physicians and basic scientists with the state-of-the-art on the "writers, readers and erasers" of poly(ADP-ribose), some recent paradigm shifts of the field and its translational potential. PMID:23727362

  19. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  20. Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

    SciTech Connect

    Kun, Ernest Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

    2008-02-08

    Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells.

  1. Synthesis and SAR of novel tricyclic quinoxalinone inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)

    SciTech Connect

    Miyashiro, Julie; Woods, Keith W.; Park, Chang H.; Liu, Xuesong; Shi, Yan; Johnson, Eric F.; Bouska, Jennifer J.; Olson, Amanda M.; Luo, Yan; Fry, Elizabeth H.; Giranda, Vincent L.; Penning, Thomas D.

    2010-09-03

    Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.

  2. Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1.

    PubMed

    Zhang, Haoyue; Xiong, Zheng-Mei; Cao, Kan

    2014-06-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria. PMID:24843141

  3. Poly (ADP-ribose) polymerase inhibitor CEP-8983 synergizes with bendamustine in chronic lymphocytic leukemia cells in vitro

    PubMed Central

    Dilley, Robert L.; Poh, Weijie; Gladstone, Douglas E.; Herman, James G.; Showel, Margaret M.; Karp, Judith E.; McDevitt, Michael A.; Pratz, Keith W.

    2014-01-01

    DNA repair aberrations and associated chromosomal instability is a feature of chronic lymphocytic leukemia (CLL). To evaluate if DNA repair insufficiencies are related to methylation changes, we examined the methylation of nine promoter regions of DNA repair proteins by bisulfide sequencing in 26 CLL primary samples and performed quantitative PCR on a subset of samples to examine BRCA1 expression. We also investigated if changes in cytogenetic or expression level of DNA repair proteins led to changes in sensitivity to a novel PARP inhibitor, CEP-8983, alone and in combination with bendamustine. No changes in promoter methylation were identified in BRCA1, BRCA2, FANC-C, FANC-F, FANC-L, ATM, MGMT, hMLH1 and H2AX except for two cases of minor BRCA1 hypermethylation. CLL samples appeared to have reduced BRCA1 mRNA expression uniformly in comparison to non-malignant lymphocytes irrespective of promoter hypermethylation. CEP-8983 displayed single agent cytotoxicity and the combination with bendamustine demonstrated synergistic cytotoxicity in the majority of CLL samples. These results were consistent across cytogenetic subgroups, including 17p deleted and previously treated patients. Our results provide rationale for further exploration of the combination of a PARP inhibitor and DNA damaging agents as a novel therapeutic strategy in CLL. PMID:24439051

  4. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  5. How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation.

    PubMed

    Mangerich, Aswin; Bürkle, Alexander

    2011-01-15

    Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by the enzyme family of poly(ADP-ribose) polymerases (PARPs). PARPs exhibit pleiotropic cellular functions ranging from maintenance of genomic stability and chromatin remodeling to regulation of cell death, thereby rendering PARP homologues promising targets in cancer therapy. Depending on the molecular status of a cancer cell, low-molecular weight PARP inhibitors can (i) either be used as monotherapeutic agents following the concept of synthetic lethality or (ii) to support classical chemotherapy or radiotherapy. The rationales are the following: (i) in cancers with selective defects in homologous recombination repair, inactivation of PARPs directly causes cell death. In cancer treatment, this phenomenon can be employed to specifically target tumor cells while sparing nonmalignant tissue. (ii) PARP inhibitors can also be used to sensitize cells to cytotoxic DNA-damaging treatments, as some PARPs actively participate in genomic maintenance. Apart from that, PARP inhibitors possess antiangiogenic functions, thus opening up a further option to inhibit tumor growth. In view of the above, a number of high-potency PARP inhibitors have been developed during the last decade and are currently evaluated as cancer therapeutics in clinical trials by several leading pharmaceutical companies. PMID:20853319

  6. Poly(ADP-Ribosyl)ation Is Required to Modulate Chromatin Changes at c-MYC Promoter during Emergence from Quiescence

    PubMed Central

    Battistelli, Cecilia; Ciotti, Agnese; Amati, Paolo; Maione, Rossella

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation. PMID:25047032

  7. ADP-Ribosyltransferases and Poly ADP-Ribosylation

    PubMed Central

    Liu, Chao; Yu, Xiaochun

    2016-01-01

    Protein ADP-ribosylation is an important posttranslational modification that plays versatile roles in multiple biological processes. ADP-ribosylation is catalyzed by a group of enzymes known as ADP-ribosyltransferases (ARTs). Using nicotinamide adenine dinucleotide (NAD+) as the donor, ARTs covalently link single or multiple ADP-ribose moieties from NAD+ to the substrates, forming mono ADP-ribosylation or poly ADP-ribosylation (PARylation). Novel functions of ARTs and ADP-ribosylation have been revealed over the past few years. Here we summarize the current knowledge on ARTs and PARylation. PMID:25938242

  8. Poly(ADP-ribosyl)ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response.

    PubMed

    Ling, Xiao Xuan; Liu, Jia Xian; Yun, Lin; DU, Yu Jun; Chen, Shao Qian; Chen, Jia Long; Tang, Huan Wen; Liu, Lin Hua

    2016-01-01

    The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation. PMID:26822515

  9. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    SciTech Connect

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-02-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

  10. Allosteric Activation of the RNF146 Ubiquitin Ligase by a Poly(ADP-ribosyl)ation Signal

    PubMed Central

    DaRosa, Paul A.; Wang, Zhizhi; Jiang, Xiaomo; Pruneda, Jonathan N.; Cong, Feng; Klevit, Rachel E.; Xu, Wenqing

    2014-01-01

    Protein poly(ADP-ribosyl)ation (PARylation) plays a role in diverse cellular processes such as DNA repair, transcription, Wnt signaling, and cell death1–6. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several critical regulatory proteins, including Axin and 3BP2 (refs 7–9). The RING-type E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is responsible for PARylation-dependent ubiquitination (PARdU)10–12. Here we provide a structural basis for RNF146 catalyzed PARdU and how PARdU specificity is achieved. First, we show that iso-ADPr, the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to efficiently bind and activate an E2. Binding of PAR/iso-ADPr induces a major conformational change that creates a functional RING structure. Thus RNF146 represents a new mechanistic class of RING E3 ligases whose activities are regulated by non-covalent ligand binding, which may provide a template for designing inducible protein-degradation systems. Second, we found that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146/TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalyzed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell. PMID:25327252

  11. Structure and function of the ARH family of ADP-ribose-acceptor hydrolases

    PubMed Central

    Mashimo, Masato; Kato, Jiro; Moss, Joel

    2014-01-01

    ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from nicotinamide adenine dinucleotide (NAD+) to specific acceptors, thereby altering their activities. The ADP-ribose transfer reactions are divided into mono- and poly-(ADP-ribosyl)ation. Cellular ADP-ribosylation levels are tightly regulated by enzymes that transfer ADP-ribose to acceptor proteins (e.g. ADP-ribosyltransferases, poly-(ADP-ribose) polymerases (PARP)) and those that cleave the linkage between ADP-ribose and acceptor (e.g. ADP-ribosyl-acceptor hydrolases (ARH), poly-(ADP-ribose) glycohydrolases (PARG)), thereby constituting an ADP-ribosylation cycle. This review summarizes current findings related to the ARH family of proteins. This family comprises three members (ARH1-3) with similar size (39 kDa) and amino acid sequence. ARH1 catalyzes the hydrolysis of the N-glycosidic bond of mono-(ADP-ribosyl)ated arginine. ARH3 hydrolyzes poly-(ADP-ribose) (PAR) and O-acetyl-ADP-ribose. The different substrate specificities of ARH1 and ARH3 contribute to their unique roles in the cell. Based on a phenotype analysis of ARH1−/− and ARH3−/− mice, ARH1 is involved in the action by bacterial toxins as well as in tumorigenesis. ARH3 participates in the degradation of PAR that is synthesized by PARP1 in response to oxidative stress-induced DNA damage; this hydrolytic reaction suppresses PAR-mediated cell death, a pathway termed parthanatos. PMID:24746921

  12. Proteomics Approaches to Identify Mono(ADP-ribosyl)ated and Poly(ADP-ribosyl)ated proteins

    PubMed Central

    Vivelo, Christina A.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  13. Proteomics approaches to identify mono-(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins.

    PubMed

    Vivelo, Christina A; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription, and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by MS using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD(+) analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  14. Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

    PubMed Central

    Blaho, J A; Michael, N; Kang, V; Aboul-Ela, N; Smulson, M E; Jacobson, M K; Roizman, B

    1992-01-01

    Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule. Images PMID:1328673

  15. Novel tricyclic poly (ADP-ribose) polymerase-1/2 inhibitors with potent anticancer chemopotentiating activity: Design, synthesis and biological evaluation.

    PubMed

    Li, Hui; Hu, Yan; Wang, Xueyan; He, Guangwei; Xu, Yungen; Zhu, Qihua

    2016-10-01

    8,9-Dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones were designed and synthesized as a new class of PARP-1/2 inhibitors. The compounds displayed a variable pattern of PARP-1/2 enzymes inhibition profile that, in part, paralleled the antiproliferative activity in cell lines. Among them, compound 9e exhibited not only the significant IC50 value of 28nM in the PARP-1 and 7.7nM in PARP-2 enzyme assay, but also a profound synergic efficacy combined with temozolomide with PF50 values of 2.6, 2.5, and 6.5 against MDA-MB-468, SW-620 and A549 and cell line, respectively. PMID:27561983

  16. DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma

    PubMed Central

    Weaver, Alice N.; Cooper, Tiffiny S.; Rodriguez, Marcela; Trummell, Hoa Q.; Bonner, James A.; Rosenthal, Eben L.; Yang, Eddy S.

    2015-01-01

    Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV− HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10–14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC. PMID:26336991

  17. Nitric Oxide (NO) Releasing Poly ADP-ribose Polymerase 1 (PARP-1) Inhibitors Targeted to Glutathione S-Transferase P1-Overexpressing Cancer Cells

    PubMed Central

    2015-01-01

    We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me2N–N(O)=NO– group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity. PMID:24521039

  18. Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase

    PubMed Central

    Moroni, F; Cozzi, A; Chiarugi, A; Formentini, L; Camaioni, E; Pellegrini-Giampietro, DE; Chen, Y; Liang, S; Zaleska, MM; Gonzales, C; Wood, A; Pellicciari, R

    2012-01-01

    BACKGROUND AND PURPOSES Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS In male rats with tMCAO, HYDAMTIQ (0.1–10 mg·kg−1) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits. PMID:21913897

  19. Inhibition of poly(ADP-ribosyl)ation in cancer: old and new paradigms revisited.

    PubMed

    Lupo, Barbara; Trusolino, Livio

    2014-08-01

    Inhibitors of poly(ADP-ribose) polymerases actualized the biological concept of synthetic lethality in the clinical practice, yielding a paradigmatic example of translational medicine. The profound sensitivity of tumors with germline BRCA mutations to PARP1/2 blockade owes to inherent defects of the BRCA-dependent homologous recombination machinery, which are unleashed by interruption of PARP DNA repair activity and lead to DNA damage overload and cell death. Conversely, aspirant BRCA-like tumors harboring somatic DNA repair dysfunctions (a vast entity of genetic and epigenetic defects known as "BRCAness") not always align with the familial counterpart and appear not to be equally sensitive to PARP inhibition. The acquisition of secondary resistance in initially responsive patients and the lack of standardized biomarkers to identify "BRCAness" pose serious threats to the clinical advance of PARP inhibitors; a feeling is also emerging that a BRCA-centered perspective might have missed the influence of additional, not negligible and DNA repair-independent PARP contributions onto therapy outcome. While regulatory approval for PARP1/2 inhibitors is still pending, novel therapeutic opportunities are sprouting from different branches of the PARP family, although they remain immature for clinical extrapolation. This review is an endeavor to provide a comprehensive appraisal of the multifaceted biology of PARPs and their evolving impact on cancer therapeutics. PMID:25026313

  20. Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly-ADP-ribosylation

    PubMed Central

    Thomas, Colin J.; Kotova, Elena; Andrake, Mark; Adolf-Bryfogle, Jared; Glaser, Robert; Regnard, Catherine; Tulin, Alexei V.

    2014-01-01

    SUMMARY Dynamically controlled post-translational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating Poly(ADP-ribose) Polymerase 1 (PARP-1). JIL-1 phosphorylates the C-terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1 dependent genes. PMID:24508391

  1. Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy

    PubMed Central

    Yang, Minghua; Liu, Liying; Xie, Min; Sun, Xiaofang; Yu, Yan; Kang, Rui; Yang, Liangchun; Zhu, Shan; Cao, Lizhi; Tang, Daolin

    2015-01-01

    Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance. PMID:25607248

  2. Nudix hydrolases degrade protein-conjugated ADP-ribose.

    PubMed

    Daniels, Casey M; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  3. Nudix hydrolases degrade protein-conjugated ADP-ribose

    PubMed Central

    Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  4. Increased poly(ADP-ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment

    PubMed Central

    Oláh, Gábor; Finnerty, Celeste; Sbrana, Elena; Elijah, Itoro; Gerö, Domokos; Herndon, David; Szabó, Csaba

    2011-01-01

    Summary Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibition. The aim of the current study was to measure the activation of PARP in human skeletal muscle biopsies at various stages of severe pediatric burn injury and to identify the cell types where this activation may occur. Another aim of the study was to test the effect of propranolol (an effective treatment of patients with burns), on the activation of PARP in skeletal muscle biopsies. PARP activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that PARP becomes activated in the skeletal muscle tissue after burns, with the peak of the activation occurring in the middle stage of the disease (13–18 days after burns). Even at the late stage of the disease (69–369 days post-burn) an elevated degree of PARP activation persisted in some of the patients. Immunohistochemical studies localized the staining of PAR primarily to vascular endothelial cells, and occasionally to resident mononuclear cells. There was a marked suppression of PARP activation in the skeletal muscle biopsies of patients who received propranolol treatment. We conclude that human burn injury is associated with the activation of PARP. We hypothesize that this response may contribute to the inflammatory responses and cell dysfunction in burns. Some of the clinical benefit of propranolol in burns may be related to its inhibitory effect on PARP activation. PMID:21368715

  5. Inhibition of poly(adenosine diphosphate-ribose) polymerase attenuates ventilator-induced lung injury

    PubMed Central

    Vaschetto, Rosanna; Kuiper, Jan W.; Chiang, Johnson; Haitsma, Jack J.; Juco, Jonathan W.; Uhlig, Stefan; Plötz, Frans B.; Della Corte, Francesco; Zhang, Haibo; Slutsky, Arthur S.

    2016-01-01

    Background Mechanical ventilation can induce organ injury associated with overwhelming inflammatory responses. Excessive activation of poly(adenosine diphosphate-ribose) polymerase enzyme following massive DNA damage may aggravate inflammatory responses. We thus hypothesized that the pharmacological inhibition of poly(adenosine diphosphate-ribose) polymerase by PJ-34 will attenuate ventilator-induced lung injury. Methods Anesthetized rats were subjected to intratracheal instillation of lipopolysaccharide at a dose of 6 mg/kg. The animals were then randomized to receive mechanical ventilation at either low tidal volume (6 mL/kg) with 5 cmH2O positive end-expiratory pressure or high tidal volume (15 mL/kg) with zero positive end-expiratory pressure, in the presence and absence of intravenous administration of PJ-34. Results The high tidal volume ventilation resulted in an increase in poly (adenosine diphosphate-ribose) polymerase activity in the lung. The treatment with PJ-34 maintained a greater oxygenation and a lower airway plateau pressure than the vehicle control group. This was associated with a decreased level of interleukin-6, active plasminogen activator inhibitor-1 in the lung, attenuated leukocyte lung transmigration and reduced pulmonary edema and apoptosis. The administration of PJ-34 also decreased the systemic levels of tumor necrosis factor-α and interleukin-6, and attenuated the degree of apoptosis in the kidney. Conclusion The pharmacological inhibition of poly(adenosine diphosphate-ribose) polymerase reduces ventilator-induced lung injury and protects kidney function. PMID:18212571

  6. Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

    PubMed

    Mangerich, Aswin; Debiak, Malgorzata; Birtel, Matthias; Ponath, Viviane; Balszuweit, Frank; Lex, Kirsten; Martello, Rita; Burckhardt-Boer, Waltraud; Strobelt, Romano; Siegert, Markus; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Bürkle, Alexander

    2016-02-26

    Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of

  7. Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

    SciTech Connect

    Kirsten, E.; Bauer, P.I.; Kun, E. San Francisco State Univ., CA )

    1991-03-01

    The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethansone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. AT a nontoxic concentration 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.

  8. Crystal Structure of Human ADP-ribose Transferase ARTD15/PARP16 Reveals a Novel Putative Regulatory Domain*

    PubMed Central

    Karlberg, Tobias; Thorsell, Ann-Gerd; Kallas, Åsa; Schüler, Herwig

    2012-01-01

    ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD+-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes. PMID:22661712

  9. Inhibition of poly(adenosine diphosphate-ribose) polymerase using quinazolinone nucleus.

    PubMed

    Hemalatha, K; Madhumitha, G

    2016-09-01

    Poly(adenosine diphosphate-ribose) polymerase (PARP) is a group of enzymes with several subtypes and it manages various ailment such as cancer, inflammatory disorders, diabetes mellitus, neuronal injury, HIV infection, Parkinsonism, aging, and ischemia-reperfusion injury. Various PARP inhibitors share a common property of bicyclic lactam in its main structural frame. The core moiety containing bicyclic lactam rings are isoquinolinones, dihydroisoquinolinones, quinazolinediones, phthalazinones, quinazolinones, and phenanthridones. The quinazolinone with diverse substituents displayed low nanomolar inhibition. Quinazolinone is an important and vital molecule in the field of medicinal chemistry possessing multitude pharmacological actions. Though the chemistry of quinazolinones has been discussed through centuries, its concise role on PARP inhibition needed a special consideration. The aim of this review is to discover the effect of quinazolinone substitutents and its role in PARP inhibition. This precise review will discuss the effect of quinazolinones on PARP subtypes such as PARP-1, PARP-2, PARP-5a, and PARP-5b. In addition to its pharmacological actions, PARP inhibitors can also act as a chemosensitizing agent, and it is used in combination with the other anticancer agents. This summarization will definitely be a supportive report for the scientist working toward the novelty in the quinazolinone nucleus and its role in PARP inhibition. PMID:27470142

  10. The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila.

    PubMed

    Wang, Zhenghan; Tian, Ai; Benchabane, Hassina; Tacchelly-Benites, Ofelia; Yang, Eungi; Nojima, Hisashi; Ahmed, Yashi

    2016-05-15

    Wnt/β-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer. PMID:27190037

  11. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    PubMed

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation. PMID:18475194

  12. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    PubMed Central

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  13. The status of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors in ovarian cancer, part 1: olaparib.

    PubMed

    Miller, Rowan E; Ledermann, Jonathan A

    2016-08-01

    Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors have shown promising clinical activity in epithelial ovarian cancer. Following the observation in vitro that PARP inhibition is synthetically lethal in tumors with BRCA mutations, PARP inhibition has become the first genotype-directed therapy for BRCA1- and BRCA2-associated ovarian cancer. However, it is becoming clear that PARP inhibition also may have clinical utility in cancers associated with defects or aberrations in DNA repair that are unrelated to BRCA mutations. Deficient DNA repair mechanisms are present in approximately 30% to 50% of high-grade serous ovarian cancers, the most common histologic subtype. Olaparib is the best-studied PARP inhibitor to date, and a number of phase 3 trials with this agent are underway. This article reviews the development of olaparib for ovarian cancer and discusses the current evidence for its use, ongoing studies, future research directions, and the challenges ahead. PMID:27487106

  14. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

    PubMed

    Sukhanova, Maria V; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M; Anarbaev, Rashid O; Curmi, Patrick A; Hamon, Loic; Lavrik, Olga I

    2016-04-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  15. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

    PubMed Central

    Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M.; Anarbaev, Rashid O.; Curmi, Patrick A.; Hamon, Loic; Lavrik, Olga I.

    2016-01-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  16. Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

    SciTech Connect

    Thraves, P.J.; Mossman, K.L.; Frazier, D.T.; Dritschilo, A.

    1986-08-01

    The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.

  17. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    SciTech Connect

    Osada, Tomoharu; Rydén, Anna-Margareta; Masutani, Mitsuko

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  18. Poly (ADP-ribose) synthetase inhibitor has a heart protective effect in a rat model of experimental sepsis

    PubMed Central

    Zhang, Lianshuang; Yao, Jinpeng; Wang, Xifeng; Li, Hongxing; Liu, Tongshen; Zhao, Wei

    2015-01-01

    The aim of this study is to investigate whether PARP inhibitor could reduce cell apoptosis and injury in the heart during sepsis. Materials and methods: 60 healthy male Sprague-Dawley (SD) rats were randomly divided into 4 groups---sham group, modal group, 3-AB pretreatment group and 3-AB treatment group, 15 rats per group. The cecal ligation and puncture (CLP) model of sepsis was used. The following were determined--levels of malondialdehyde (MDA), ATP and nicotinamide adenine dinucleotide (NAD+), expression of PARP, Bcl-2, Bax, cytochrome C and caspase 3 activity in the myocardium tissue, levels of serum creatine kinase muscle brain (CK-MB) fraction and troponin I. Results: Histological and molecular analyses showed that myocardial cells apoptosis were associated with mitochondria injury, with an increase in the amount of PARP and a decrease in ATP and NAD+ levels in model group. In addition, the levels of Bax, cytochrome C and caspase 3 activity, serum levels of CK-MB and troponin I increased, but levels of Bcl-2 significantly decreased. Inhibition of PARP upregulated the levels of ATP, NAD + and Bcl-2, and significantly reduced the activation of PARP and caspase 3, decreased the levels of MDA, cytochrome C, CK-MB and troponin I. As a result, apoptosis in the heart was attenuated. Conclusion: These results indicate that PARP activation may be involved in apoptosis in the heart induced by sepsis and 3-AB could improve it. PMID:26617692

  19. Structural biology of the writers, readers, and erasers in mono- and poly(ADP-ribose) mediated signaling

    PubMed Central

    Karlberg, Tobias; Langelier, Marie-France; Pascal, John M.; Schüler, Herwig

    2013-01-01

    ADP-ribosylation of proteins regulates protein activities in various processes including transcription control, chromatin organization, organelle assembly, protein degradation, and DNA repair. Modulating the proteins involved in the metabolism of ADP-ribosylation can have therapeutic benefits in various disease states. Protein crystal structures can help understand the biological functions, facilitate detailed analysis of single residues, as well as provide a basis for development of small molecule effectors. Here we review recent advances in our understanding of the structural biology of the writers, readers, and erasers of ADP-ribosylation. PMID:23458732

  20. The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei

    PubMed Central

    Haines, M. E.; Johnston, I. R.; Mathias, A. P.; Ridge, D.

    1969-01-01

    1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed. PMID:4311824

  1. Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase.

    PubMed

    Wang, Zhenghan; Tacchelly-Benites, Ofelia; Yang, Eungi; Thorne, Curtis A; Nojima, Hisashi; Lee, Ethan; Ahmed, Yashi

    2016-05-01

    Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation. PMID:26975665

  2. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    SciTech Connect

    Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

    2014-02-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

  3. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

    SciTech Connect

    Komissarova, Elena V.; Rossman, Toby G.

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  4. ADP-ribose-derived nuclear ATP synthesis by NUDIX5 is required for chromatin remodeling.

    PubMed

    Wright, Roni H G; Lioutas, Antonios; Le Dily, Francois; Soronellas, Daniel; Pohl, Andy; Bonet, Jaume; Nacht, A S; Samino, Sara; Font-Mateu, Jofre; Vicent, Guillermo P; Wierer, Michael; Trabado, Miriam A; Schelhorn, Constanze; Carolis, Carlo; Macias, Maria J; Yanes, Oscar; Oliva, Baldo; Beato, Miguel

    2016-06-01

    Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation. PMID:27257257

  5. Fanconi anemia protein FANCD2 inhibits TRF1 polyADP-ribosylation through tankyrase1-dependent manner

    PubMed Central

    2011-01-01

    Background Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres. Results We reveal that FANCD2 may perform a novel function separate to the FANCD2/BRCA pathway. This function includes FANCD2 interaction with one of the telomere components, the PARP family member tankyrase-1. Moreover, FANCD2 inhibits tankyrase-1 activity in vitro. In turn, FANCD2 deficiency increases the polyADP-ribosylation of telomere binding factor TRF1. Conclusions FANCD2 binding and inhibiting tankyrase-1PARsylation at telomeres may provide an additional step within the FA pathway for the regulation of genomic integrity. PMID:21314979

  6. DNA polymerase β-dependent cell survival independent of XRCC1 expression.

    PubMed

    Horton, Julie K; Gassman, Natalie R; Dunigan, Brittany D; Stefanick, Donna F; Wilson, Samuel H

    2015-02-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  7. DNA polymerase β-dependent cell survival independent of XRCC1 expression

    PubMed Central

    Horton, Julie K.; Gassman, Natalie R.; Dunigan, Brittany B.; Stefanick, Donna F.; Wilson, Samuel H.

    2014-01-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1+/+ and Xrcc1−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β- dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  8. Pre-exposure to heat shock inhibits peroxynitrite-induced activation of poly(ADP) ribosyltransferase and protects against peroxynitrite cytotoxicity in J774 macrophages.

    PubMed

    Szabó, C; Wong, H R; Salzman, A L

    1996-11-14

    The reaction of nitric oxide (NO) with superoxide yields the cytotoxic oxidant peroxynitrite, produced during inflammation and shock. A novel pathway of peroxynitrite cytotoxicity involves activation of the nuclear enzyme poly(ADP) ribosyltransferase, and concomitant ADP-ribosylation. NAD+ consumption and exhaustion of intracellular energy stores. In the present report we provide evidence that pre-exposure of J774 macrophages to heat shock reduces peroxynitrite-induced activation of poly(ADP) ribosyltransferase and protects against the peroxynitrite-induced suppression of mitochondrial respiration. The protection was significant at 8 h after heat shock, but was absent at 24 h after heat shock. Thus, the protection showed a temporal correlation with the expression of heat shock protein 70, the expression of which was maximal at 8 h. Exposure to heat shock did not alter the expression of poly(ADP) ribosyltransferase over 24 h. In summary, the heat shock phenotype or heat shock proteins may protect against peroxynitrite induced cytotoxicity. PMID:8960887

  9. NIH study uncovers new mechanism of action for class of chemotherapy drugs

    Cancer.gov

    NIH researchers have discovered a significant new mechanism of action for a class of chemotherapy drugs known as poly (ADP-ribose) polymerase inhibitors, or PARP inhibitors. They have also identified differences in the toxic capabilities of three drugs in

  10. Recent advances in the Okamoto model: the CD38-cyclic ADP-ribose signal system and the regenerating gene protein (Reg)-Reg receptor system in beta-cells.

    PubMed

    Okamoto, Hiroshi; Takasawa, Shin

    2002-12-01

    Twenty years ago, we first proposed our hypothesis on beta-cell damage and its prevention (the Okamoto model), according to which poly(ADP-ribose) synthetase/polymerase (PARP) activation is critically involved in the consumption of NAD(+), leading to energy depletion and cell death by necrosis. Recently, the model was reconfirmed by results using PARP knockout mice and has been recognized as providing the basis for necrotic death of various cells and tissues. Based on the model, we proposed two signal systems in beta-cells: one is the CD38-cyclic ADP-ribose (cADPR) signal system for insulin secretion, and the other is the regenerating gene protein (Reg)-Reg receptor system for beta-cell regeneration. The physiological and pathological significance of the two signal systems in a variety of cells and tissues as well as in pancreatic beta-cells has recently been recognized. Here, we describe the Okamoto model and its descendents, the CD38-cADPR signal system and the Reg-Reg receptor system, focusing on recent advances and how their significance came to light. Because PARP is involved in Reg gene transcription to induce beta-cell regeneration, and the PARP activation reduces the cellular NAD(+) to decrease the formation of cADPR (a second messenger for insulin secretion) and further to cause necrotic beta-cell death, PARP and its inhibitors have key roles in the induction of beta-cell regeneration, the maintenance of insulin secretion, and the prevention of beta-cell death. PMID:12475791

  11. Profile of olaparib in the treatment of advanced ovarian cancer

    PubMed Central

    Chase, Dana M; Patel, Shreya; Shields, Kristin

    2016-01-01

    Olaparib is a poly(ADP-ribose) polymerase inhibitor that received accelerated approval from the US Food and Drug Administration as monotherapy for patients with germline BRCA mutations and ovarian cancer treated with three or more prior lines of chemotherapy. This article summarizes the mechanism of poly(ADP-ribose) polymerase inhibition, therapeutic profile and uses of olaparib, and current and ongoing literature pertaining to olaparib in advanced ovarian cancer. PMID:27186080

  12. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    SciTech Connect

    Neuhof, Dirk Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-11-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.

  13. Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis.

    PubMed

    Yu, Wenxi; Zhang, Zhichang; Min, Daliu; Yang, Qingcheng; Du, Xuefei; Tang, Lina; Lin, Feng; Sun, Yuanjue; Zhao, Hui; Zheng, Shuier; He, Aina; Li, Hongtao; Yao, Yang; Shen, Zan

    2014-04-01

    Osteosarcoma (OS) is the most common primary malignant tumour of bone. Nearly 30-40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumourigenesis and progression of OS is crucial for developing new therapies. Here, we found that the expression of Mediator of RNA polymerase II transcription subunit 19 (Med19) was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis (P<0.05) and poor prognosis (P<0.01). Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP). In addition, Med19 knockdown significantly suppressed tumour growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 downregulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumour samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumour metastasis in a model of OS metastatic spreading. Taken together, our data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway. PMID:24565852

  14. Involvement of PARP1 in the regulation of alternative splicing

    PubMed Central

    Matveeva, Elena; Maiorano, John; Zhang, Qingyang; Eteleeb, Abdallah M; Convertini, Paolo; Chen, Jing; Infantino, Vittoria; Stamm, Stefan; Wang, Jiping; Rouchka, Eric C; Fondufe-Mittendorf, Yvonne N

    2016-01-01

    Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes. PMID:27462443

  15. Cytological and molecular studies of chromosomal radiosensitivity in Down Syndrome cells

    SciTech Connect

    MacLaren, R.A.

    1988-01-01

    Molecular, cellular and cytogenetic studies were conducted to determine if altered levels of poly(ADP-ribose) polymerase, a DNA repair-related enzyme, is responsible for the reported formation of excess X-ray induced chromosome aberrations in cells derived from Down Syndrome (DS) patients. Nonstimulated lymphocytes from normal and DS subjects were pretreated with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, for 30 minutes before exposure to X-rays and the levels of induced chromosome aberrations were determined in mitotic cells. DS lymphocytes exhibited significantly higher frequencies of chromosome aberrations in the presence of 3-aminobenzamide that normal lymphocytes. No difference was observed in the absence of 3-aminobenzamide. Additional studies were done using normal and DS lymphoblastoid cell lines which exhibited a similar response at the DNA level as the lymphocytes. Analysis of poly(ADP-ribose) polymerase activity based on incorporation of the substrate, NAD{sup +}, into acid insoluble materials, revealed that there was no significant difference in the ability to form poly (ADP-ribose) in the DS or normal cells. 3-aminobenzamide effectively inhibited poly(ADP-ribose) polymerase in both the normal and DS cells.

  16. New PARP targets for cancer therapy

    PubMed Central

    Vyas, Sejal; Chang, Paul

    2015-01-01

    Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD+ as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5a, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, the majority of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer relevant functions for these PARPs, indicating that we need to understand more about these PARPs in order to target them effectively. PMID:24898058

  17. Olaparib.

    PubMed

    Goulooze, S C; Cohen, A F; Rissmann, R

    2016-01-01

    Olaparib is used to treat BReast CAncer susceptibility protein (BRCA)-associated, platinum-sensitive ovarian cancer. Olaparib inhibits poly(ADP-ribose) polymerase, thereby blocking the repair of single-strand DNA breaks. This results in synthetic lethality in BRCA-associated cancer cells, which have a dysfunction of another DNA repair pathway - homologous recombination. PMID:26344419

  18. Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.

    PubMed

    McLick, J; Hakam, A; Bauer, P I; Kun, E; Zacharias, D E; Glusker, J P

    1987-06-01

    The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide

  19. Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

    PubMed Central

    Hassa, Paul O.; Haenni, Sandra S.; Elser, Michael; Hottiger, Michael O.

    2006-01-01

    Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD+-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as “programmed necrosis” (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., “histone code”), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear

  20. Mitochondrial and Nuclear Cross Talk in Cell Death: Parthanatos

    PubMed Central

    Andrabi, Shaida A.; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) PARP-1 is an abundant nuclear protein first described to facilitate DNA base excision repair. Recent work has expanded the physiologic functions of PARP-1 and it is clear that the full range of biologic actions of this important protein are not yet fully understood. Regulation of the product of PARP-1, poly(ADP-ribose) (PAR), is a dynamic process with poly(ADP-ribose) glycohydrolase (PARG) playing a major role in the degradation of the polymer. Under pathophysiologic situations, over activation of poly(ADP-ribose) polymerase-1 (PARP-1) results in unregulated PAR synthesis and widespread neuronal cell death. Once thought to be necrotic cell death due to energy failure, we recently found that PARP-1 dependent cell death is dependent on the generation of PAR that triggers nuclear translocation of apoptosis-inducing factor (AIF) to result in caspase-independent cell death. This form of cell death is distinct from apoptosis, necrosis or autophagy and is termed Parthanatos. PARP-1 dependent cell death has been implicated in tissues throughout the body and in diseases afflicting hundreds of millions world wide including stroke, Parkinson's disease, heart attack, diabetes, and ischemia reperfusion injury in numerous tissues. The breadth of indications for PARP-1 injury make Parthanatos a clinically important form of cell death to understand and control. PMID:19076445

  1. ADP-Ribosylation: Activation, Recognition, and Removal

    PubMed Central

    Li, Nan; Chen, Junjie

    2014-01-01

    ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions. PMID:24552704

  2. PARP1 Val762Ala polymorphism reduces enzymatic activity

    SciTech Connect

    Wang Xiaogan; Wang Zhaoqi; Tong Weimin . E-mail: tong@iarc.fr; Shen Yan

    2007-03-02

    Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K {sub m} of PARP1-Ala762 was increased to a 1.2-fold of the K {sub m} of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K {sub m}. This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism.

  3. Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

    PubMed

    Debiak, Malgorzata; Lex, Kirsten; Ponath, Viviane; Burckhardt-Boer, Waltraud; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Mangerich, Aswin; Bürkle, Alexander

    2016-02-26

    Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the

  4. PARP is involved in replicative aging in Neurospora crassa

    PubMed Central

    Kothe, Gregory O.; Kitamura, Maki; Masutani, Mitsuko; Selker, Eric U.; Inoue, Hirokazu

    2010-01-01

    Modification of proteins by the addition of poly(ADP-ribose) is carried out by poly(ADP-ribose) polymerases (PARPs). PARPs have been implicated in a wide range of biological processes in eukaryotes, but no universal function has been established. A study of the Aspergillus nidulans PARP ortholog (PrpA) revealed that the protein is essential and involved in DNA repair, reminiscent of findings using mammalian systems. We found that a Neurospora PARP orthologue (NPO) is dispensable for cell survival, DNA repair and epigenetic silencing but that replicative aging of mycelia is accelerated in an npo mutant strain. We propose that PARPs may control aging as proposed for Sirtuins, which also consume NAD+ and function either as mono(ADP-ribose) transferases or protein deacetylases. PARPs may regulate aging by impacting NAD+/NAM availability, thereby influencing Sirtuin activity, or they may function in alternative NAD+-dependent or NAD+-independent aging pathways. PMID:20045739

  5. Enantioselective synthesis of tetrafluorinated ribose and fructose.

    PubMed

    Linclau, Bruno; Boydell, A James; Timofte, Roxana S; Brown, Kylie J; Vinader, Victoria; Weymouth-Wilson, Alexander C

    2009-02-21

    A perfluoroalkylidene lithium mediated cyclisation approach for the enantioselective synthesis of a tetrafluorinated aldose (ribose) and of a tetrafluorinated ketose (fructose), both in the furanose and in the pyranose form, is described. PMID:19194597

  6. Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

    PubMed Central

    2010-01-01

    Background Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism. PMID:20388209

  7. Induction of apoptosis in MCF-7 cells by β-1,3-xylooligosaccharides prepared from Caulerpa lentillifera.

    PubMed

    Maeda, Reiko; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

    2012-01-01

    β-1,3-Xylan was prepared from the green alga, Caulerpa lentillifera, and hydrolyzed to oligosaccharides by a mild acid treatment. The average degree of polymerization was about 5. The oligosaccharides reduced the number of viable human breast cancer MCF-7 cells in a dose-dependent manner, and induced chromatin condensation and degradation of poly ADP-ribose polymerase, indicating that they induced apoptosis in MCF-7 cells. PMID:22738982

  8. Nicotinamide and the skin.

    PubMed

    Chen, Andrew C; Damian, Diona L

    2014-08-01

    Nicotinamide, an amide form of vitamin B3, boosts cellular energy and regulates poly-ADP-ribose-polymerase 1, an enzyme with important roles in DNA repair and the expression of inflammatory cytokines. Nicotinamide shows promise for the treatment of a wide range of dermatological conditions, including autoimmune blistering disorders, acne, rosacea, ageing skin and atopic dermatitis. In particular, recent studies have also shown it to be a potential agent for reducing actinic keratoses and preventing skin cancers. PMID:24635573

  9. Detecting and quantifying pADPr in vivo.

    PubMed

    Lai, Yi-Chen; Aneja, Rajesh K; Satchell, Margaret A; Clark, Robert S B

    2011-01-01

    Poly(ADP-ribose) polymerases (PARP) participate in diverse biological processes contributing to cellular homeostasis or exacerbating injury. PARP catalyzes the addition of ADP-ribose molecules (pADPr) to the target proteins, a process termed poly-ADP-ribosylation. Overactivation of PARP, as reflected by increased poly-ADP-ribosylation, accumulation of pADPr-modified proteins or free pADPr, contributes to the depletion of NAD(+) and mitochondrial dysfunction, potentially leading to cell death via apoptosis or necrosis. Since PARP over-activation has been identified as a key contributor to the pathobiology of many diseases, monitoring PARP 1 activation by detecting and quantifying pADPr may provide valuable mechanistic insights as well as facilitating therapeutic drug monitoring for PARP inhibitors.Several non-isotopic immunodetection methods for quantifying pADPr are discussed: western blotting of poly-ADP-ribosylated proteins, cellular localization of pADPr by immunohistochemistry, quantification of pADPr by enzyme-linked immunoassay and small-scale two-dimensional gel electrophoresis. PMID:21870258

  10. (-)-Botryodiplodin, A Unique Ribose Analog Toxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many toxins owe their mechanisms of action to being structural analogs of essential metabolites, messengers or structural components. Examples range from tubo-curare to penicillin. Ribose plays a unique role in the metabolism of living organisms, whether prokaryotes or eukaryotes. It and its deri...

  11. The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

    PubMed

    Altmeyer, Matthias; Toledo, Luis; Gudjonsson, Thorkell; Grøfte, Merete; Rask, Maj-Britt; Lukas, Claudia; Akimov, Vyacheslav; Blagoev, Blagoy; Bartek, Jiri; Lukas, Jiri

    2013-10-24

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling. PMID:24055346

  12. Helicobacter pylori activation of PARP-1

    PubMed Central

    Nossa, Carlos W

    2010-01-01

    Chronic infection of the human stomach by Helicobacter pylori is an important risk factor for gastric cancer. H. pylori produces a cache of virulence factors that promote colonization and persistence, which, in turn, contributes to a robust inflammatory response at the host-pathogen interface. Recently, we reported that H. pylori activates the abundant nuclear regulator poly(ADP-ribose) polymerase (PARP)-1, resulting in the production of the catabolite poly(ADP-ribose) (PAR). PARP-1 is emerging as a key player in establishing homeostasis at the host-pathogen interface. In this article, we summarize the discovery of H. pylori-dependent PARP-1 activation, and discuss potential roles for PARP-1 in H. pylori-mediated gastric disease. In light of the remarkable successes that have reported for treating inflammatory disorders and cancers with PARP-1 inhibitors, we discuss the prospects of targeting PARP-1 for treatment of H. pylori-associated gastric disease. PMID:21468218

  13. Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

    PubMed Central

    Berger, N A; Berger, S J; Catino, D M; Petzold, S J; Robins, R K

    1985-01-01

    Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents. PMID:3919063

  14. Synthesis of phosphonate and phostone analogues of ribose-1-phosphates

    PubMed Central

    Nasomjai, Pitak; Slawin, Alexandra M Z

    2009-01-01

    Summary The synthesis of phosphonate analogues of ribose-1-phosphate and 5-fluoro-5-deoxyribose-1-phosphate is described. Preparations of both the α- and β-phosphonate anomers are reported for the ribose and 5-fluoro-5-deoxyribose series and a synthesis of the corresponding cyclic phostones of each α-ribose is also reported. These compounds have been prepared as tools to probe the details of fluorometabolism in S. cattleya. PMID:19777136

  15. Coordination of DNA repair by NEIL1 and PARP-1: a possible link to aging

    PubMed Central

    Noren Hooten, Nicole; Fitzpatrick, Megan; Kompaniez, Kari; Jacob, Kimberly D.; Moore, Brittany R.; Nagle, Julia; Barnes, Janice; Lohani, Althaf; Evans, Michele K.

    2012-01-01

    Oxidative DNA damage accumulates with age and is repaired primarily via the base excision repair (BER) pathway. This process is initiated by DNA glycosylases, which remove damaged bases in a substrate-specific manner. The DNA glycosylases human 8-oxoguanine-DNA glycosylase (OGG1) and NEIL1, a mammalian homolog of Escherichia coli endonuclease VIII, have overlapping yet distinct substrate specificity. Recently, we reported that OGG1 binds to the Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein that poly(ADP-ribosyl)ates nuclear proteins in response to DNA damage and other cellular signals. Here, we show that NEIL1 and PARP-1 bind both in vitro and in vivo. PARP-1 binds to the C-terminal-100 amino acids of NEIL1 and NEIL1 binds to the BRCT domain of PARP-1. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. Furthermore, NEIL-deficient fibroblasts have impaired poly(ADP-ribosyl)ation of cellular proteins after DNA damage, which can be rescued by NEIL1 expression. Additionally, PARP-1 inhibits NEIL1 incision activity in a concentration-dependent manner. Consistent with the idea of impaired DNA repair during aging, we observed differential binding of PARP-1 to recombinant NEIL1 in older mice compared to younger mice. These data further support the idea that dynamic interplay between different base excision repair proteins is important for efficient BER. PMID:23104860

  16. Dielectric relaxation study of the dynamics of monosaccharides: D-ribose and 2-deoxy-D-ribose

    NASA Astrophysics Data System (ADS)

    Kaminski, K.; Kaminska, E.; Wlodarczyk, P.; Paluch, M.; Ziolo, J.; Ngai, K. L.

    2008-08-01

    The dielectric loss spectra of two closely related monosaccharides, D-ribose and 2-deoxy-D-ribose, measured at ambient and elevated pressures are presented. 2-deoxy-D-ribose and D-ribose are respectively the building blocks of the backbone chains in the nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Small differences in the structure between D-ribose and 2-deoxy-D-ribose result in changes of the glass transition temperature Tg, as well as the dielectric strength and activation enthalpy of the secondary relaxations. However, the frequency dispersion of the structural α-relaxation for the same relaxation time remains practically the same. Two secondary relaxations are present in both sugars. The slower secondary relaxation shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the slower secondary relaxation is the important and 'universal' Johari-Goldstein β-relaxation of both sugars according to one of the criteria set up to classify secondary relaxations. Additional confirmation of this conclusion comes from good agreement of the observed relaxation time of the slower secondary relaxation with the primitive relaxation time calculated from the coupling model. All the dynamic properties of D-ribose and 2-deoxy-D-ribose are similar to the other monosaccharides, glucose, fructose, galactose and sorbose, except for the much larger relaxation strength of the α-relaxation of the former compared to the latter. The difference may distinguish the chemical and biological functions of D-ribose and 2-deoxy-D-ribose from the other monosaccharides.

  17. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

    PubMed

    Gagné, Jean-Philippe; Ethier, Chantal; Defoy, Daniel; Bourassa, Sylvie; Langelier, Marie-France; Riccio, Amanda A; Pascal, John M; Moon, Kyung-Mee; Foster, Leonard J; Ning, Zhibin; Figeys, Daniel; Droit, Arnaud; Poirier, Guy G

    2015-06-01

    An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process. PMID:25800440

  18. Ribose Accelerates Gut Motility and Suppresses Mouse Body Weight Gaining

    PubMed Central

    Liu, Yan; Li, Tong-Ruei R; Xu, Cong; Xu, Tian

    2016-01-01

    The increasing prevalence of obesity is closely related to excessive energy consumption. Clinical intervention of energy intake is an attractive strategy to fight obesity. However, the current FDA-approved weight-loss drugs all have significant side effects. Here we show that ribose upregulates gut motility and suppresses mice body weight gain. Ribokinase, which is encoded by Rbks gene, is the first enzyme for ribose metabolism in vivo. Rbks mutation resulted in ribose accumulation in the small intestine, which accelerated gut movement. Ribose oral treatment in wild type mice also enhanced bowel motility and rendered mice resistance to high fat diets. The suppressed weight gain was resulted from enhanced ingested food excretion. In addition, the effective dose of ribose didn't cause any known side effects (i.e. diarrhea and hypoglycemia). Overall, our results show that ribose can regulate gut motility and energy homeostasis in mice, and suggest that administration of ribose and its analogs could regulate gastrointestinal motility, providing a novel therapeutic approach for gastrointestinal dysfunction and weight control. PMID:27194947

  19. Synergistic inhibition of Streptococcal biofilm by ribose and xylitol.

    PubMed

    Lee, Heon-Jin; Kim, Se Chul; Kim, Jinkyung; Do, Aejin; Han, Se Yeong; Lee, Bhumgey David; Lee, Hyun Ho; Lee, Min Chan; Lee, So Hui; Oh, Taejun; Park, Sangbin; Hong, Su-Hyung

    2015-02-01

    Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol. PMID:25463908

  20. The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis.

    PubMed

    Wachsman, J T

    1996-02-19

    There is compelling evidence for the central role of oxidative damage in the aging process and for the participation of reactive oxygen species in tumor initiation and promotion. Caloric restriction (CR) or energy restriction retards age-associated increases in mitochondrial free-radical production and reduces the accumulation of oxidatively damaged cell components. CR has also been shown to slow down age-related declines in various repair capabilities, including some types of DNA repair. It is proposed that inhibitors of mitochondrial electron transport and/or uncouplers of oxidative phosphorylation (rotenone, amytal, amiodarone, valinomycin, etc.), when used at extremely low doses, could mimic the effects of CR in model systems. The objective is to lower mitochondrial free-radical production by decreasing the fraction of electron carriers in the reduced state. In addition to a variety of other effects, CR has been shown to increase the rate of apoptosis, particularly in preneoplastic cells, and in general, to promote elevated levels of free glucocorticoids (GCs). GCs are known to induce tissue-specific apoptosis and to upregulate gap-junction-mediated intercellular communication (GJIC). Tumor promoters like phorbol esters have the opposite effect, in that they inhibit both the process of apoptosis and GJIC. The enzyme poly (ADP-ribose) polymerase (PARP) is thought to play a central role in apoptosis, in a manner that has been highly conserved in evolution. There is good evidence that the apoptosis-associated Ca/Mg-dependent DNA endonuclease is maintained in a latent form by being poly (ADP-ribosylated). Apoptosis would require the removal of this polymer from the endonuclease, and, most likely, its removal from topoisomerase II and histone H1 as well. The role of poly (ADP-ribose) in apoptosis, carcinogenesis, and aging could be studied by the use of modulators of PARP activity (3-aminobenzamide, 3-nitrosobenzamide, 1% ethanol, etc.), inhibitors of poly ADP-ribose

  1. Selective derivatization and sequestration of ribose from a prebiotic mix.

    PubMed

    Springsteen, Greg; Joyce, Gerald F

    2004-08-11

    Observations regarding the catalytic potential of RNA and the role of RNA in biology have formed the basis for the "RNA world" hypothesis, which suggests that a genetic system based on self-replicating polyribonucleotides preceded modern biology. However, attempts to devise a realistic prebiotic synthesis of nucleic acids from simple starting materials have been plagued by problems of poor chemical selectivity, lack of stereo- and regiospecificity, and similar rates of formation and degradation of some of the key intermediates. For example, ribose would have been only a small component of a highly complex mix of sugars resulting from the condensation of formaldehyde in a prebiotic world. In addition, ribose is more reactive and degrades more rapidly compared with most other monosaccharides. This study demonstrates an approach for the preferential sequestration of ribose relative to other sugars that takes advantage of its greater reactivity. Cyanamide reacts especially rapidly with ribose to form a stable bicyclic adduct. This product crystallizes spontaneously in aqueous solution, whereas the corresponding products derived from threose, galactose, glucose, mannose, and each of the other pentoses do not. Furthermore, when employing a racemic mixture of d- and l-ribose, enantiomerically twinned crystals are formed that contain discrete homochiral domains. PMID:15291561

  2. Chromatin to clinic: The molecular rationale for PARP1 inhibitor function

    PubMed Central

    Knudsen, Karen E.; de Bono, Johann S.; Rubin, Mark A.; Feng, Felix Y.

    2015-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) inhibitors were recently shown to have clinical impact in a number of disease settings, particularly as related to cancer therapy, treatment for cardiovascular dysfunction, and suppression of inflammation. The molecular basis for PARP1 inhibitor function is complex, and appears to depend on the dual roles of PARP1 in DNA damage repair and transcriptional regulation. Here, the mechanisms by which PARP-1 inhibitors elicit clinical response are discussed, and strategies for translating the preclinical elucidation of PARP-1 function into advances in disease management are reviewed. PMID:26091341

  3. SIRT6 promotes DNA repair under stress by activating PARP1.

    PubMed

    Mao, Zhiyong; Hine, Christopher; Tian, Xiao; Van Meter, Michael; Au, Matthew; Vaidya, Amita; Seluanov, Andrei; Gorbunova, Vera

    2011-06-17

    Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)-ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress. PMID:21680843

  4. A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

    PubMed

    Calderwood, Stuart K

    2016-05-26

    Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

  5. Novel PARP-1 inhibitors based on a 2-propanoyl-3H-quinazolin-4-one scaffold.

    PubMed

    Giannini, Giuseppe; Battistuzzi, Gianfranco; Vesci, Loredana; Milazzo, Ferdinando M; De Paolis, Francesca; Barbarino, Marcella; Guglielmi, Mario Berardino; Carollo, Valeria; Gallo, Grazia; Artali, Roberto; Dallavalle, Sabrina

    2014-01-15

    Poly(ADP-ribose)polymerase-I (PARP-1) enzyme is involved in maintaining DNA integrity and programmed cell death. A virtual screening of commercial libraries led to the identification of five novel scaffolds with inhibitory profile in the low nanomolar range. A hit-to-lead optimization led to the identification of a group of new potent PARP-1 inhibitors, acyl-piperazinylamides of 3-(4-oxo-3,4-dihydro-quinazolin-2-yl)-propionic acid. Molecular modeling studies highlighted the preponderant role of the propanoyl side chain. PMID:24388690

  6. Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Park, Cheon-Soo; Ahn, Yongchel; Lee, Dahae; Moon, Sung Won; Kim, Ki Hyun; Yamabe, Noriko; Hwang, Gwi Seo; Jang, Hyuk Jai; Lee, Heesu; Kang, Ki Sung; Lee, Jae Wook

    2015-12-15

    Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway. PMID:26564263

  7. Olaparib for the treatment of ovarian cancer.

    PubMed

    Bornstein, E; Jimeno, A

    2016-01-01

    Olaparib, an oral poly(ADP-ribose) polymerase (PARP) inhibitor, is the first FDA-approved drug in its class for patients with ovarian cancer, specifically in a subset of patients with BRCA mutations and prior chemotherapy treatments. PARP inhibitors have had other implications in different solid tumor types including breast, gastric and pancreatic malignancies. In light of the recent FDA approval of olaparib for the treatment of ovarian cancer, this article aims to outline the mechanisms and implications of the drug. With a favorable adverse event profile and improved outcomes, including progression-free survival, olaparib has demonstrated augmentation to therapeutic options in the treatment of ovarian cancer. PMID:26937492

  8. New Strategies in Ewings Sarcoma: Lost in Translation?

    PubMed Central

    Arnaldez, Fernanda I.; Helman, Lee J.

    2014-01-01

    Ewings Sarcoma is the second most common pediatric malignant bone tumor. Aggressive multimodality therapy has led to an improvement in outcomes, particularly in patients with localized disease. However, therapy-related toxicities are non trivial and the prognosis for patients with relapsed and/or metastatic disease continues to be poor. In this article we outline some of the promising therapies that have the potential to change the Ewings Sarcoma therapeutic paradigm in the not too distant future: insulin growth factor receptor inhibitors, targeting of the fusion protein, epigenetic manipulation, poly-ADP-ribose polymerase inhibitors and immunotherapy. PMID:24756371

  9. Homologous recombination deficiency: Exploiting the fundamental vulnerability of ovarian cancer

    PubMed Central

    Konstantinopoulos, Panagiotis A.; Ceccaldi, Raphael; Shapiro, Geoffrey I.; D’Andrea, Alan D.

    2015-01-01

    Approximately 50% of epithelial ovarian cancers (EOCs) exhibit defective DNA repair via homologous recombination (HR) due to genetic and epigenetic alterations of HR pathway genes. Defective HR is an important therapeutic target in EOC as exemplified by the efficacy of platinum analogues in this disease, as well as the advent of poly-ADP ribose polymerase inhibitors which exhibit synthetic lethality when applied to HR deficient cells. Here, we describe the genotypic and phenotypic characteristics of HR deficient EOCs, discuss current and emerging approaches for targeting these tumors, and present challenges associated with these approaches focusing on development and overcoming resistance. PMID:26463832

  10. RIBOSE MODIFIED NUCLEOSIDES AND NUCLEOTIDES AS LIGANDS FOR PURINE RECEPTORS

    PubMed Central

    Ravi, R. G.; Nandanan, E.; Kim, H. S.; Moro, S.; Kim, Y. C.; Lee, K.; Barak, D.; Marquez, V. E.; Ji, X. D.

    2016-01-01

    Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3′,5′-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs. PMID:11563046

  11. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  12. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  13. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  14. Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment

    ERIC Educational Resources Information Center

    Simon, Erin; Cook, Katie; Pritchard, Meredith R.; Stripe, Wayne; Bruch, Martha; Bendinskas, Kestutis

    2010-01-01

    This exercise provides students hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, proton nuclear magnetic resonance ([superscript 1]H NMR) spectroscopy, and kinetic and thermodynamic product formation. In this laboratory experiment, the methyl acetal of ribose is synthesized, and the kinetic and thermodynamic…

  15. NAD+ metabolism in health and disease.

    PubMed

    Belenky, Peter; Bogan, Katrina L; Brenner, Charles

    2007-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid. PMID:17161604

  16. PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway

    PubMed Central

    Kishi, Yuta; Fujihara, Hisako; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Fujihara, Yuko; Hamada, Yoshiki; Satomura, Kazuhito; Masutani, Mitsuko

    2015-01-01

    Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels. PMID:26492236

  17. PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway.

    PubMed

    Kishi, Yuta; Fujihara, Hisako; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Fujihara, Yuko; Hamada, Yoshiki; Satomura, Kazuhito; Masutani, Mitsuko

    2015-01-01

    Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels. PMID:26492236

  18. Cockayne syndrome group B protein (CSB) plays a general role in chromatin maintenance and remodeling

    PubMed Central

    Newman, John C.; Bailey, Arnold D.; Weiner, Alan M.

    2006-01-01

    Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways. PMID:16772382

  19. Eukaryotic TLS polymerases.

    PubMed

    Tomczyk, Przemysław; Synowiec, Ewelina; Wysokiński, Daniel; Woźniak, Katarzyna

    2016-01-01

    TLS polymerases are able to replicate damaged DNA (called translesion DNA synthesis, TLS). Their presence prevents cell death as a result of violating the integrity of the genome. In vitro, they are mutator, but in vivo are recruited by specific types of DNA damage and usually replicate them in a correct manner. The best-known TLS polymerases belong to the Y family, such as Rev1, κ, η, ι, and polymerase ζ from the B family. There are two mechanisms of TLS polymerases action: polymerase-switching model and the gap-filling model. Selection of the mechanism primarily depends on the phase of the cell cycle. The regulation of these polymerases may take place at the transcriptional level and at level of recruitment to the sites of DNA damage. In the latter case post-translational modification of proteins - ubiquitination and sumoylation, and protein-protein interactions are crucial. PMID:27333922

  20. The Juxtaposition of Ribose Hydroxyl Groups: The Root of Biological Catalysis and the RNA World?

    NASA Astrophysics Data System (ADS)

    Bernhardt, Harold S.

    2015-06-01

    We normally think of enzymes as being proteins; however, the RNA world hypothesis suggests that the earliest biological catalysts may have been composed of RNA. One of the oldest surviving RNA enzymes we are aware of is the peptidyl transferase centre (PTC) of the large ribosomal RNA, which joins amino acids together to form proteins. Recent evidence indicates that the enzymatic activity of the PTC is principally due to ribose 2 '-OHs. Many other reactions catalyzed by RNA and/or in which RNA is a substrate similarly utilize ribose 2 '-OHs, including phosphoryl transfer reactions that involve the cleavage and/or ligation of the ribose-phosphate backbone. It has recently been proposed by Yakhnin (2013) that phosphoryl transfer reactions were important in the prebiotic chemical evolution of RNA, by enabling macromolecules composed of polyols joined by phosphodiester linkages to undergo recombination reactions, with the reaction energy supplied by the phosphodiester bond itself. The almost unique juxtaposition of the ribose 2'-hydroxyl and 3'-oxygen in ribose-containing polymers such as RNA, which gives ribose the ability to catalyze such reactions, may have been an important factor in the selection of ribose as a component of the first biopolymer. In addition, the juxtaposition of hydroxyl groups in free ribose: (i) allows coordination of borate ions, which could have provided significant and preferential stabilization of ribose in a prebiotic environment; and (ii) enhances the rate of permeation by ribose into a variety of lipid membrane systems, possibly favouring its incorporation into early metabolic pathways and an ancestral ribose-phosphate polymer. Somewhat more speculatively, hydrogen bonds formed by juxtaposed ribose hydroxyl groups may have stabilized an ancestral ribose-phosphate polymer against degradation (Bernhardt and Sandwick 2014). I propose that the almost unique juxtaposition of ribose hydroxyl groups constitutes the root of both biological

  1. Bioenergetic Impairment in Animal and Cellular Models of Alzheimer's Disease: PARP-1 Inhibition Rescues Metabolic Dysfunctions.

    PubMed

    Martire, Sara; Fuso, Andrea; Mosca, Luciana; Forte, Elena; Correani, Virginia; Fontana, Mario; Scarpa, Sigfrido; Maras, Bruno; d'Erme, Maria

    2016-08-10

    Amyloid-beta peptide accumulation in the brain is one of the main hallmarks of Alzheimer's disease. The amyloid aggregation process is associated with the generation of free radical species responsible for mitochondrial impairment and DNA damage that in turn activates poly(ADP-ribose)polymerase 1 (PARP-1). PARP-1 catalyzes the poly(ADP-ribosylation), a post-translational modification of proteins, cleaving the substrate NAD+ and transferring the ADP-ribose moieties to the enzyme itself or to an acceptor protein to form branched polymers of ADP-ribose. In this paper, we demonstrate that a mitochondrial dysfunction occurs in Alzheimer's transgenic mice TgCRND8, in SH-SY5Y treated with amyloid-beta and in 7PA2 cells. Moreover, PARP-1 activation contributes to the functional energetic decline affecting cytochrome oxidase IV protein levels, oxygen consumption rates, and membrane potential, resulting in cellular bioenergetic deficit. We also observed, for the first time, an increase of pyruvate kinase 2 expression, suggesting a modulation of the glycolytic pathway by PARP-1. PARP-1 inhibitors are able to restore both mitochondrial impairment and pyruvate kinase 2 expression. The overall data here presented indicate a pivotal role for this enzyme in the bioenergetic network of neuronal cells and open new perspectives for investigating molecular mechanisms underlying energy charge decline in Alzheimer's disease. In this scenario, PARP-1 inhibitors might represent a novel therapeutic intervention to rescue cellular energetic metabolism. PMID:27567805

  2. Escherchia coli ribose binding protein based bioreporters revisited

    PubMed Central

    Reimer, Artur; Yagur-Kroll, Sharon; Belkin, Shimshon; Roy, Shantanu; van der Meer, Jan Roelof

    2014-01-01

    Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system. PMID:25005019

  3. Global transcriptome response in Lactobacillus sakei during growth on ribose

    PubMed Central

    2011-01-01

    Background Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. Results The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved. Conclusions Our data shows that the ribose uptake and catabolic machinery in

  4. Viola plant cyclotide vigno 5 induces mitochondria-mediated apoptosis via cytochrome C release and caspases activation in cervical cancer cells.

    PubMed

    Esmaeili, Mohammad Ali; Abagheri-Mahabadi, Nazanin; Hashempour, Hossein; Farhadpour, Mohsen; Gruber, Christian W; Ghassempour, Alireza

    2016-03-01

    Cyclotides describe a unique cyclic peptide family that displays a broad range of biological activities including uterotonic, anti-bacteria, anti-cancer and anti-HIV. The vigno cyclotides consist of vigno 1-10 were reported recently from Viola ignobilis. In the present study, we examined the effects of vigno 5, a natural cyclopeptide from V. ignobilis, on cervical cancer cells and the underlying mechanisms. We found that vigno 5-treated Hela cells were killed off by apoptosis in a dose-dependent manner within 24h, and were characterized by the appearance of nuclear shrinkage, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. The mitochondrial pathway of apoptosis revealed that cytochrome C is released from mitochondria to cytosol, associated with the activation of caspase-9 and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP). Overall, the results indicate that vigno 5 induces apoptosis in part via the mitochondrial pathway, which is associated with a release of cytochrome C and elevated activity of caspase-9 and -3 in Hela cells. PMID:26751970

  5. Cellular regulation of ADP-ribosylation of proteins: 3. Selective augmentation of in vitro ADP-ribosylation of histone H3 in murine thymic cells after in vivo emetine treatment

    SciTech Connect

    Sooki-Toth, A.; Banfalvi, G.; Staub, M.; Antoni, F. ); Szoelloesi, J. ); Kirsten, E. ); Kun, E. )

    1989-09-01

    Thymic cells were isolated at intervals of between 0 and 144 h from mice that received one intraperitoneal injection of emetine, and thymus weight, incorporation of ({sup 14}C)leucine into proteins and ({sup 3}H)thymidine into DNA in intact thymic cells, as well as initial rates of protein ADP-ribosylation in permeabilized cells were simultaneously monitored. The effect of emetine as an inhibitor of protein synthesis corresponds to the induction of sequential cellular events, such as cell exit and remigration, by other antimitotic agents and produces an activation of proliferation of cells reentering into this organ. Proliferation, as demonstrated by a large increase in DNA synthesis and entrance into S phase, was kinetically related to an apparent increase in poly (ADP-ribose) polymerase activity in thymic cells and a highly significant in vitro ADP-ribosylation of histone H3. since no DNA fragmentation occurred in thymic cells, as tested by a fluorometric technique it is probable that a selective activation of poly (ADP-ribose) polymerase may have been induced in cells that undergo differentiation and proliferation while repopulating thymus.

  6. Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

    PubMed Central

    Bognar, Eszter; Sarszegi, Zsolt; Szabo, Aliz; Debreceni, Balazs; Kalman, Nikoletta; Tucsek, Zsuzsanna; Sumegi, Balazs; Gallyas, Ferenc

    2013-01-01

    Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease. PMID:23755222

  7. Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

    PubMed

    Zheng, Chun-Lei; Sumizawa, Tomoyuki; Che, Xiao-Fang; Tsuyama, Shinichiro; Furukawa, Tatsuhiko; Haraguchi, Misako; Gao, Hui; Gotanda, Takenari; Jueng, Hei-Cheul; Murata, Fusayoshi; Akiyama, Shin-Ichi

    2005-01-01

    Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly. PMID:15567158

  8. Ataxia telangiectasia mutated (ATM) is dispensable for endonuclease I-SceI-induced homologous recombination in mouse embryonic stem cells.

    PubMed

    Rass, Emilie; Chandramouly, Gurushankar; Zha, Shan; Alt, Frederick W; Xie, Anyong

    2013-03-01

    Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency. PMID:23355489

  9. 1,1-Bis(3′-indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits bladder cancer growth

    PubMed Central

    Inamoto, Teruo; Papineni, Sabitha; Chintharlapalli, Sudhakar; Cho, Sung-Dae; Safe, Stephen; Kamat, Ashish M.

    2016-01-01

    Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor I-B subfamily of transcription factors with no known endogenous ligand or stimulator. We show, for the first time, evidence that Nurr1 is expressed in a panel of 11 human bladder cancer cell lines. A new class of methylene-substituted diindolylmethanes (C-DIM) were screened and 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)-methane (DIM-C-pPhCl) activated the ligand-binding domain of Nurr1. Treatment of bladder cancer cells with Nurr1-active C-DIM resulted in decreased cell survival (MTT assay) and induction of cell death pathways, resulting in poly(ADP-ribose) polymerase cleavage and DNA fragmentation. The specificity of the Nurr1-active compound was shown using RNA interference in 253J B-V cells, whereby small interfering RNA against Nurr1 attenuated ligand-dependent activation of Nurr1 and poly(ADP-ribose) polymerase cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand and small interfering RNA experiments attenuated tumor necrosis factor-related apoptosis-inducing ligand production. In an orthotopic model of human bladder tumors established in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a novel agent for activating Nurr1. PMID:19074857

  10. Diabetic Neuropathy and Oxidative Stress: Therapeutic Perspectives

    PubMed Central

    Hosseini, Asieh; Abdollahi, Mohammad

    2013-01-01

    Diabetic neuropathy (DN) is a widespread disabling disorder comprising peripheral nerves' damage. DN develops on a background of hyperglycemia and an entangled metabolic imbalance, mainly oxidative stress. The majority of related pathways like polyol, advanced glycation end products, poly-ADP-ribose polymerase, hexosamine, and protein kinase c all originated from initial oxidative stress. To date, no absolute cure for DN has been defined; although some drugs are conventionally used, much more can be found if all pathophysiological links with oxidative stress would be taken into account. In this paper, although current therapies for DN have been reviewed, we have mainly focused on the links between DN and oxidative stress and therapies on the horizon, such as inhibitors of protein kinase C, aldose reductase, and advanced glycation. With reference to oxidative stress and the related pathways, the following new drugs are under study such as taurine, acetyl-L-carnitine, alpha lipoic acid, protein kinase C inhibitor (ruboxistaurin), aldose reductase inhibitors (fidarestat, epalrestat, ranirestat), advanced glycation end product inhibitors (benfotiamine, aspirin, aminoguanidine), the hexosamine pathway inhibitor (benfotiamine), inhibitor of poly ADP-ribose polymerase (nicotinamide), and angiotensin-converting enzyme inhibitor (trandolapril). The development of modern drugs to treat DN is a real challenge and needs intensive long-term comparative trials. PMID:23738033

  11. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

    SciTech Connect

    Shirai, Hidenori; Fujimori, Hiroaki; Gunji, Akemi; Maeda, Daisuke; Hirai, Takahisa; Poetsch, Anna R.; Harada, Hiromi; Yoshida, Tomoko; Sasai, Keisuke; Okayasu, Ryuichi; Masutani, Mitsuko

    2013-05-24

    Highlights: •Parg{sup −/−} ES cells were more sensitive to γ-irradiation than Parp-1{sup −/−} ES cells. •Parg{sup −/−} cells were more sensitive to carbon-ion irradiation than Parp-1{sup −/−} cells. •Parg{sup −/−} cells showed defects in DSB repair after carbon-ion irradiation. •PAR accumulation was enhanced after carbon-ion irradiation compared to γ-irradiation. -- Abstract: Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg{sup −/−} and poly(ADP-ribose) polymerase-1 deficient (Parp-1{sup −/−}) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg{sup −/−} cells were more sensitive to γ-irradiation than Parp-1{sup −/−} cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg{sup −/−} cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg{sup −/−} ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1{sup −/−} cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg{sup −/−} ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg{sup −/−} cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1{sup −/−} cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24 h. The induction level of p53 phophorylation at ser18 was

  12. Nucleolar Integrity Is Required for the Maintenance of Long-Term Synaptic Plasticity

    PubMed Central

    Allen, Kim D.; Gourov, Andrei V.; Harte, Christopher; Gao, Peng; Lee, Clarice; Sylvain, Darlene; Splett, Joshua M.; Oxberry, William C.; van de Nes, Paula S.; Troy-Regier, Matthew J.; Wolk, Jason; Alarcon, Juan M.; Hernández, A. Iván

    2014-01-01

    Long-term memory (LTM) formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose) polymerase-1 (PARP-1), are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP) in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosyl)ation (PAR) dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP) ribose molecules (pADPr) after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA) and extracellular signal-regulated kinase (ERK)—two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I), responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components)—hence, new ribosomes and nucleoli integrity—are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation. PMID:25089620

  13. TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1

    PubMed Central

    Zou, Lian-Hong; Shang, Zeng-Fu; Tan, Wei; Liu, Xiao-Dan; Xu, Qin-Zhi; Song, Man; Wang, Yu; Guan, Hua; Zhang, Shi-Meng; Yu, Lan; Zhong, Cai-Gao; Zhou, Ping-Kun

    2015-01-01

    TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs. PMID:25749521

  14. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain.

    PubMed

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  15. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain

    PubMed Central

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  16. Rates of Decomposition of Ribose and other Sugars: Implications for Chemical Evolution

    NASA Technical Reports Server (NTRS)

    Larralde, Rosa; Robertson, Michael P.; Miller, Stanley L.

    1995-01-01

    The existence of the RNA world, in which RNA acted as a catalyst as well as an informational macromolecule, assumes a large prebiotic source of ribose or the existence of pre-RNA molecules with backbones different from ribose-phosphate. The generally accepted prebiotic synthesis of ribose, the formose reaction, yields numerous sugars without any selectivity. Even if there were a selective synthesis of ribose, there is still the problem of stability. Sugars are known to be unstable in strong acid or base, but there are few data for neutral solutions. Therefore, we have measured the rate of decomposition of ribose between pH 4 and pH 8 from 40 C to 120 C. The ribose half-lives are very short (73 min at pH 7.0 and 100 C and 44 years at pH 7.0 and 0 C). The other aldopentoses and aldohexoses have half-lives within an order of magnitude of these values, as do 2-deoxyribose, ribose 5-phosphate, and ribose 2,4bisphosphate. These results suggest that the backbone of the first genetic material could not have contained ribose or other sugars because of their instability.

  17. Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

    PubMed Central

    Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos

    2015-01-01

    Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type. PMID:25692488

  18. Abscisic acid signaling through cyclic ADP-ribose in plants

    SciTech Connect

    Wu, Yan; Kuzma, J.; Marechal, E.

    1997-12-19

    Abscisic acid (ABA) is the primary hormone that mediates plant responses to stresses such as cold, drought, and salinity. Single-cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction. Cyclic ADP-ribose (cADPR) was identified as a signaling molecule in the ABA response and was shown to exert its effects by way of calcium. Bioassay experiments showed that the amounts of cADPR in Arabidopsis thaliana plants increased in response to ABA treatment and before ABA-induced gene expression.

  19. Transcriptional Control by PARP-1: Chromatin Modulation, Enhancer-binding, Coregulation, and Insulation

    PubMed Central

    Kraus, W. Lee

    2008-01-01

    Summary The regulation of gene expression requires a wide array of protein factors that can modulate chromatin structure, act at enhancers, function as transcriptional coregulators, or regulate insulator function. Poly(ADP-ribose) polymerase-1 (PARP-1), an abundant and ubiquitous nuclear enzyme that catalyzes the NAD+-dependent addition of ADP-ribose polymers on a variety of nuclear proteins, has been implicated in all of these functions. Recent biochemical, genomic, proteomic, and cell-based studies have highlighted the role of PARP-1 in each of these processes and provided new insights about the molecular mechanisms governing PARP-1-dependent regulation of gene expression. In addition, these studies have demonstrated how PARP-1 functions as an integral part of cellular signaling pathways that culminate in gene regulatory outcomes. PMID:18450439

  20. Role of PARP-1 in prostate cancer

    PubMed Central

    Deshmukh, Dhanraj; Qiu, Yun

    2015-01-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) is an enzyme that catalyzes the covalent attachment of polymers of ADP-ribose (PAR) moieties on itself and its target proteins. PARP1 activity is frequently deregulated in various cancers and therefore it has emerged as a new drug target for cancer therapy. The role of PARP-1 in DNA repair has been well documented and BRCA mutations are implicated for determining the sensitivity to PARP inhibitors. Recent studies also point to a role of PARP-1 in transcription regulation which may contribute to oncogenic signaling and cancer progression. Given that efficacy of PARP inhibitors are also seen in patients not harboring BRCA mutations, some other mechanisms might also be involved. In the present review, we highlight the mechanisms by which PARP-1 regulates gene expression in prostate cancer and provide an overview of the ongoing clinical trials using PARP inhibitors in various cancers including prostate cancer. PMID:26069882

  1. [Cancer therapy by PARP inhibitors].

    PubMed

    Seimiya, Hiroyuki

    2015-08-01

    Poly(ADP-ribose) polymerases(PARP) synthesize the ADP-ribose polymers onto proteins and play a role in DNA repair. PARP inhibitors block the repair of single-strand breaks, which in turn gives rise to double-strand breaks during DNA replication. Thus, PARP inhibitors elicit synthetic lethality in cancer with BRCA1/2 loss-of-function mutations that hamper homologous recombination repair of double-strand breaks. Olaparib, the first-in-class PARP inhibitor, was approved for treatment of BRCA-mutated ovarian cancer in Europe and the United States in 2014. Other PARP inhibitors under clinical trials include rucaparib, niraparib, veliparib, and the "PARP-trapping" BMN-673. BRCA1/2 sequencing is an FDA-approved companion diagnostics, which predicts the cancer vulnerability to PARP inhibition. Together, synthetic lethal PARP inhibition is a novel promising strategy for cancer intervention even in cases without prominent driver oncogenes. PMID:26281686

  2. Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs.

    PubMed

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J

    2016-04-01

    Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3 Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments. PMID:27124456

  3. Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs

    NASA Astrophysics Data System (ADS)

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V.; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J.

    2016-04-01

    Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3. Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments.

  4. Impact of template backbone heterogeneity on RNA polymerase II transcription

    PubMed Central

    Xu, Liang; Wang, Wei; Zhang, Lu; Chong, Jenny; Huang, Xuhui; Wang, Dong

    2015-01-01

    Variations in the sugar component (ribose or deoxyribose) and the nature of the phosphodiester linkage (3′-5′ or 2′-5′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eukaryotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity remain elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quantitatively investigated pol II transcriptional behaviors through different template backbone variants. We revealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phosphodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA. PMID:25662224

  5. Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors

    PubMed Central

    Sahaboglu, A; Tanimoto, N; Bolz, S; Garrido, M G; Ueffing, M; Seeliger, M W; Löwenheim, H; Ekström, P; Paquet-Durand, F

    2014-01-01

    Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD. PMID:24853412

  6. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  7. TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation.

    PubMed

    Yonezawa, Ryo; Yamamoto, Shinichiro; Takenaka, Miki; Kage, Yukiko; Negoro, Takaharu; Toda, Takahiro; Ohbayashi, Masayuki; Numata, Tomohiro; Nakano, Yasuko; Yamamoto, Toshinori; Mori, Yasuo; Ishii, Masakazu; Shimizu, Shunichi

    2016-01-01

    Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation. PMID:26600069

  8. The role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells.

    PubMed

    Wang, Hui; Lu, Changqing; Li, Qing; Xie, Jun; Chen, Tongbing; Tan, Yan; Wu, Changping; Jiang, Jingting

    2014-11-01

    This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1. PMID:25377255

  9. Elemental bioimaging of Cisplatin in Caenorhabditis elegans by LA-ICP-MS

    PubMed Central

    Crone, Barbara; Aschner, Michael; Schwerdtle, Tanja; Karst, Uwe; Bornhorst, Julia

    2015-01-01

    Cis-diamminedichloroplatinum(II) (Cisplatin) is one of the most important and frequently used cytostatic drugs for the treatment of various solid tumors. Herein, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method incorporating a fast and simple sample preparation protocol was developed for the elemental mapping of Cisplatin in the model organism Caenorhabditis elegans (C. elegans). The method allows imaging of the spatially-resolved elemental distribution of platinum in the whole organism with respect to the anatomic structure in L4 stage worms at a lateral resolution of 5 µm. In addition, a dose- and time-dependent Cisplatin uptake was corroborated quantitatively by a total reflection X-ray fluorescence spectroscopy (TXRF) method, and the elemental mapping indicated that Cisplatin is located in the intestine and in the head of the worms. Better understanding of the distribution of Cisplatin in this well-established model organism will be instrumental in deciphering Cisplatin toxicity and pharmacokinetics. Since the cytostatic effect of Cisplatin is based on binding the DNA by forming intra- and interstrand crosslinks, the response of poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants to Cisplatin was also examined. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase 1 (PARP-1) led to disturbed DNA damage response. With respect to survival and brood size, pme-1 deletion mutants were more sensitive to Cisplatin as compared to wildtype worms, while Cisplatin uptake was indistinguishable. PMID:25996669

  10. Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

    SciTech Connect

    Negi, Geeta; Kumar, Ashutosh; Sharma, Shyam S.

    2010-01-01

    Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).

  11. Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    2002-09-24

    Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

  12. PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

    PubMed

    Luijsterburg, Martijn S; de Krijger, Inge; Wiegant, Wouter W; Shah, Rashmi G; Smeenk, Godelieve; de Groot, Anton J L; Pines, Alex; Vertegaal, Alfred C O; Jacobs, Jacqueline J L; Shah, Girish M; van Attikum, Haico

    2016-02-18

    The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability. PMID:26895424

  13. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    PubMed

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  14. Basal activity of a PARP1-NuA4 complex varies dramatically across cancer cell lines

    PubMed Central

    Krukenberg, Kristin A.; Jiang, Ruomu; Steen, Judith A.; Mitchison, Timothy J.

    2014-01-01

    Summary Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important post-translational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed a new assay for measuring PARP activity in cell lysates, and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high and low activity lines. A newly discovered complex containing the NuA4 chromatin remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a new pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA damage response. PMID:25199834

  15. Pleiotropic Cellular Functions of PARP1 in Longevity and Aging: Genome Maintenance Meets Inflammation

    PubMed Central

    Mangerich, Aswin; Bürkle, Alexander

    2012-01-01

    Aging is a multifactorial process that depends on diverse molecular and cellular mechanisms, such as genome maintenance and inflammation. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1), which catalyzes the synthesis of the biopolymer poly(ADP-ribose), exhibits an essential role in both processes. On the one hand, PARP1 serves as a genomic caretaker as it participates in chromatin remodelling, DNA repair, telomere maintenance, resolution of replicative stress, and cell cycle control. On the other hand, PARP1 acts as a mediator of inflammation due to its function as a regulator of NF-κB and other transcription factors and its potential to induce cell death. Consequently, PARP1 represents an interesting player in several aging mechanisms and is discussed as a longevity assurance factor on the one hand and an aging-promoting factor on the other hand. Here, we review the molecular mechanisms underlying the various roles of PARP1 in longevity and aging with special emphasis on cellular studies and we briefly discuss the results in the context of in vivo studies in mice and humans. PMID:23050038

  16. Cytokine-mediated β-cell damage in PARP-1-deficient islets

    PubMed Central

    Meares, Gordon P.; Hughes, Katherine J.; Hansen, Polly A.; Corbett, John A.

    2012-01-01

    Poly(ADP)-ribose polymerase (PARP) is an abundant nuclear protein that is activated by DNA damage; once active, it modifies nuclear proteins through attachment of poly(ADP)-ribose units derived from β-nicotinamide adenine dinucleotide (NAD+). In mice, the deletion of PARP-1 attenuates tissue injury in a number of animal models of human disease, including streptozotocin-induced diabetes. Also, inflammatory cell signaling and inflammatory gene expression are attenuated in macrophages isolated from endotoxin-treated PARP-1-deficient mice. In this study, the effects of PARP-1 deletion on cytokine-mediated β-cell damage and macrophage activation were evaluated. There are no defects in inflammatory mediator signaling or inflammatory gene expression in macrophages and islets isolated from PARP-1-deficient mice. While PARP-1 deficiency protects islets against cytokine-induced islet cell death as measured by biochemical assays of membrane polarization, the genetic absence of PARP-1 does not effect cytokine-induced inhibition of insulin secretion or cytokine-induced DNA damage in islets. While PARP-1 deficiency appears to provide protection from cell death, it fails to provide protection against the inhibitory actions of cytokines on insulin secretion or the damaging actions on islet DNA integrity. PMID:22535743

  17. cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function.

    PubMed

    Jaumann, Mirko; Dettling, Juliane; Gubelt, Martin; Zimmermann, Ulrike; Gerling, Andrea; Paquet-Durand, François; Feil, Susanne; Wolpert, Stephan; Franz, Christoph; Varakina, Ksenya; Xiong, Hao; Brandt, Niels; Kuhn, Stephanie; Geisler, Hyun-Soon; Rohbock, Karin; Ruth, Peter; Schlossmann, Jens; Hütter, Joachim; Sandner, Peter; Feil, Robert; Engel, Jutta; Knipper, Marlies; Rüttiger, Lukas

    2012-02-01

    Noise-induced hearing loss (NIHL) is a global health hazard with considerable pathophysiological and social consequences that has no effective treatment. In the heart, lung and other organs, cyclic guanosine monophosphate (cGMP) facilitates protective processes in response to traumatic events. We therefore analyzed NIHL in mice with a genetic deletion of the gene encoding cGMP-dependent protein kinase type I (Prkg1) and found a greater vulnerability to and markedly less recovery from NIHL in these mice as compared to mice without the deletion. Prkg1 was expressed in the sensory cells and neurons of the inner ear of wild-type mice, and its expression partly overlapped with the expression profile of cGMP-hydrolyzing phosphodiesterase 5 (Pde5). Treatment of rats and wild-type mice with the Pde5 inhibitor vardenafil almost completely prevented NIHL and caused a Prkg1-dependent upregulation of poly (ADP-ribose) in hair cells and the spiral ganglion, suggesting an endogenous protective cGMP-Prkg1 signaling pathway that culminates in the activation of poly (ADP-ribose) polymerase. These data suggest vardenafil or related drugs as possible candidates for the treatment of NIHL. PMID:22270721

  18. Is There an Epigenetic Component Underlying the Resistance of Triple-Negative Breast Cancers to Parp Inhibitors?

    PubMed Central

    Lovato, Amanda; Panasci, Lawrence; Witcher, Michael

    2012-01-01

    Poly(ADP-ribose) polymerase (Parp) is an enzyme responsible for catalyzing post-translational modifications through the addition of poly(ADP-ribose) chains (known as PARylation). Modification by PARylation modulates numerous cellular processes including transcription, chromatin remodeling, apoptosis, and DNA damage repair. In particular, the role of Parp activation in response to DNA damage has been intensely studied. Tumors bearing mutations of the breast cancer susceptibility genes, Brca1/2, are prone to DNA breakages whose restoration into functional double-strand DNA is Parp dependent. This concept has been exploited therapeutically in Brca mutated breast and ovarian tumors, where acute sensitivity to Parp inhibitors is observed. Based on in vitro and clinical studies it remains unclear to what extent Parp inhibitors can be utilized beyond treating Brca mutated tumors. This review will focus on the often overlooked roles of PARylation in chromatin remodeling, epigenetics, and transcription to explain why some cancers may be unresponsive to Parp inhibition. We predict that understanding the impact of PARylation on gene expression will lead to alternative approaches to manipulate the Parp pathway for therapeutic benefit. PMID:23293602

  19. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells. PMID:27064363

  20. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities

    PubMed Central

    Fatokun, Amos A; Dawson, Valina L; Dawson, Ted M

    2014-01-01

    Cells die by a variety of mechanisms. Terminally differentiated cells such as neurones die in a variety of disorders, in part, via parthanatos, a process dependent on the activity of poly (ADP-ribose)-polymerase (PARP). Parthanatos does not require the mediation of caspases for its execution, but is clearly mechanistically dependent on the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor (AIF). The nuclear translocation of this otherwise beneficial mitochondrial protein, occasioned by poly (ADP-ribose) (PAR) produced through PARP overactivation, causes large-scale DNA fragmentation and chromatin condensation, leading to cell death. This review describes the multistep course of parthanatos and its dependence on PAR signalling and nuclear AIF translocation. The review also discusses potential targets in the parthanatos cascade as promising avenues for the development of novel, disease-modifying, therapeutic agents. LINKED ARTICLES This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24684389

  1. Parthanatos, a messenger of death.

    PubMed

    David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

    2009-01-01

    Poly-ADP-ribose polymerase-1 (PARP-1)'s roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 overactivation underlies cell death in models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into understanding mechanisms downstream of PARP-1 overactivation. Recent evidence shows that poly-ADP ribose (PAR) polymer itself can act as a cell death effector downstream of PARP-1. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will present evidence and questions raised by these recent findings, and summarize the proposed mechanisms by which PARP-1 overactivation kills. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 overactivation. PMID:19273119

  2. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin

    PubMed Central

    Robert, Carine; Nagaria, Pratik K.; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J.; Cole, Philip A.; Rassool, Feyruz V.

    2016-01-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP “trapping”. Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP “trapping”, which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells. PMID:27064363

  3. PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining

    PubMed Central

    Luijsterburg, Martijn S.; de Krijger, Inge; Wiegant, Wouter W.; Shah, Rashmi G.; Smeenk, Godelieve; de Groot, Anton J.L.; Pines, Alex; Vertegaal, Alfred C.O.; Jacobs, Jacqueline J.L.; Shah, Girish M.; van Attikum, Haico

    2016-01-01

    Summary The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability. PMID:26895424

  4. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1

    PubMed Central

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E.; Riccio, Amanda A.; Pascal, John M.; Neuhaus, David

    2015-01-01

    Summary Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins. PMID:26626479

  5. The Elephant and the Blind Men: Making Sense of PARP Inhibitors in Homologous Recombination Deficient Tumor Cells.

    PubMed

    De Lorenzo, Silvana B; Patel, Anand G; Hurley, Rachel M; Kaufmann, Scott H

    2013-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is an important component of the base excision repair (BER) pathway as well as a regulator of homologous recombination (HR) and non-homologous end-joining (NHEJ). Previous studies have demonstrated that treatment of HR-deficient cells with PARP inhibitors results in stalled and collapsed replication forks. Consequently, HR-deficient cells are extremely sensitive to PARP inhibitors. Several explanations have been advanced to explain this so-called synthetic lethality between HR deficiency and PARP inhibition: (i) reduction of BER activity leading to enhanced DNA double-strand breaks, which accumulate in the absence of HR; (ii) trapping of inhibited PARP1 at sites of DNA damage, which prevents access of other repair proteins; (iii) failure to initiate HR by poly(ADP-ribose) polymer-dependent BRCA1 recruitment; and (iv) activation of the NHEJ pathway, which selectively induces error-prone repair in HR-deficient cells. Here we review evidence regarding these various explanations for the ability of PARP inhibitors to selectively kill HR-deficient cancer cells and discuss their potential implications. PMID:24062981

  6. Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

    PubMed Central

    Sahaboglu, Ayse; Kranz, Katharina; Michalakis, Stylianos; Farinelli, Pietro; Koch, Susanne; Koch, Fred; Cottet, Sandra; Janssen-Bienhold, Ulrike; Dedek, Karin; Biel, Martin; Zrenner, Eberhart; Euler, Thomas; Ekström, Per; Ueffing, Marius; Paquet-Durand, François

    2014-01-01

    Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments. PMID:25392995

  7. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  8. Polymerase chain displacement reaction.

    PubMed

    Harris, Claire L; Sanchez-Vargas, Irma J; Olson, Ken E; Alphey, Luke; Fu, Guoliang

    2013-02-01

    Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics. PMID:23384180

  9. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

    PubMed Central

    Negm, Fayek B.; Marlow, Gary C.

    1985-01-01

    This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID:16664320

  10. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  11. Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein.

    PubMed

    Liu, Chien-Hung; Zhou, Ligang; Chen, Guifang; Krug, Robert M

    2015-11-10

    Previous studies showed that ZAPL (PARP-13.1) exerts its antiviral activity via its N-terminal zinc fingers that bind the mRNAs of some viruses, leading to mRNA degradation. Here we identify a different antiviral activity of ZAPL that is directed against influenza A virus. This ZAPL antiviral activity involves its C-terminal PARP domain, which binds the viral PB2 and PA polymerase proteins, leading to their proteasomal degradation. After the PB2 and PA proteins are poly(ADP-ribosylated), they are associated with the region of ZAPL that includes both the PARP domain and the adjacent WWE domain that is known to bind poly(ADP-ribose) chains. These ZAPL-associated PB2 and PA proteins are then ubiquitinated, followed by proteasomal degradation. This antiviral activity is counteracted by the viral PB1 polymerase protein, which binds close to the PARP domain and causes PB2 and PA to dissociate from ZAPL and escape degradation, explaining why ZAPL only moderately inhibits influenza A virus replication. Hence influenza A virus has partially won the battle against this newly identified ZAPL antiviral activity. Eliminating PB1 binding to ZAPL would be expected to substantially increase the inhibition of influenza A virus replication, so that the PB1 interface with ZAPL is a potential target for antiviral development. PMID:26504237

  12. Biochemical preparation of L-ribose and L-arabinose from ribitol: a new approach.

    PubMed

    Ahmed, Z; Shimonishi, T; Bhuiyan, S H; Utamura, M; Takada, G; Izumori, K

    1999-01-01

    L-ribose and L-arabinose were prepared biochemically from ribitol via a two-step reaction, by which the complete oxidation of ribitol to L-ribulose (approximately 98%) was achieved by the reaction of washed cells of Acetobacter aceti IFO 3281. The produced L-ribulose was then used as a substrate for the production of L-ribose and L-arabinose. The isomerization of L-ribulose to L-ribose and L-arabinose was carried out using L-ribose isomerase (L-RI) of Acinetobacter sp. strain DL-28 and L-arabinose isomerase (L-AI) of Mycobacterium smegmatis, respectively. At equilibrium, the ratio of L-ribose: L-ribulose was 70:30 and that of L-arabinose: L-ribulose was 90: 10. After a simple purification treatment, both pentoses could be crystallized without the use of column chromatography. The crystals were confirmed as L-ribose and L-arabinose by High-performance liquid chromatography (HPLC), Infrared (IR), Nuclear magnetic resonance (NMR) and optical rotation measurements. PMID:16232643

  13. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    PubMed Central

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  14. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

    PubMed

    McInerney, Peter; Adams, Paul; Hadi, Masood Z

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  15. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGESBeta

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  16. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    PubMed Central

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  17. Mouse models of DNA polymerases.

    PubMed

    Menezes, Miriam R; Sweasy, Joann B

    2012-12-01

    In 1956, Arthur Kornberg discovered the mechanism of the biological synthesis of DNA and was awarded the Nobel Prize in Physiology or Medicine in 1959 for this contribution, which included the isolation and characterization of Escherichia coli DNA polymerase I. Now there are 15 known DNA polymerases in mammalian cells that belong to four different families. These DNA polymerases function in many different cellular processes including DNA replication, DNA repair, and damage tolerance. Several biochemical and cell biological studies have provoked a further investigation of DNA polymerase function using mouse models in which polymerase genes have been altered using gene-targeting techniques. The phenotypes of mice harboring mutant alleles reveal the prominent role of DNA polymerases in embryogenesis, prevention of premature aging, and cancer suppression. PMID:23001998

  18. Polymerase Activity of Pichinde Virus

    PubMed Central

    Carter, Michael F.; Biswal, Nilambar; Rawls, William E.

    1974-01-01

    Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg2+ and Mn2+ were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients. PMID:4132669

  19. Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase*

    PubMed Central

    Kasiviswanathan, Rajesh; Copeland, William C.

    2011-01-01

    During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2′-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3–14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3′-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed. PMID:21778232

  20. Hyperthermal (1-100 eV) nitrogen ion scattering damage to D-ribose and 2-deoxy-D-ribose films.

    PubMed

    Deng, Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A

    2007-10-14

    Highly charged heavy ion traversal of a biological medium can produce energetic secondary fragment ions. These fragment ions can in turn cause collisional and reactive scattering damage to DNA. Here we report hyperthermal (1-100 eV) scattering of one such fragment ion (N(+)) from biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose condensed on polycrystalline Pt substrate. The results indicate that N(+) ion scattering at kinetic energies down to 10 eV induces effective decomposition of both sugar molecules and leads to the desorption of abundant cation and anion fragments. Use of isotope-labeled molecules (5-(13)C D-ribose and 1-D D-ribose) partly reveals some site specificity of the fragment origin. Several scattering reactions are also observed. Both ionic and neutral nitrogen atoms abstract carbon from the molecules to form CN(-) anion at energies down to approximately 5 eV. N(+) ions also abstract hydrogen from hydroxyl groups of the molecules to form NH(-) and NH(2) (-) anions. A fraction of OO(-) fragments abstract hydrogen to form OH(-). The formation of H(3)O(+) ions also involves hydrogen abstraction as well as intramolecular proton transfer. These findings suggest a variety of severe damaging pathways to DNA molecules which occur on the picosecond time scale following heavy ion irradiation of a cell, and prior to the late diffusion-limited homogeneous chemical processes. PMID:17935431

  1. Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae

    PubMed Central

    Yang, Jun; Sharma, Sunny; Kötter, Peter; Entian, Karl-Dieter

    2015-01-01

    Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications. PMID:25653162

  2. Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae.

    PubMed

    Yang, Jun; Sharma, Sunny; Kötter, Peter; Entian, Karl-Dieter

    2015-02-27

    Methylation of ribose sugars at the 2'-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2'-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5' central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D' box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications. PMID:25653162

  3. Neuronal trauma model: in search of Thanatos.

    PubMed

    Cole, Kasie; Perez-Polo, J Regino

    2004-11-01

    Trauma to the nervous system triggers responses that include oxidative stress due to the generation of reactive oxygen species (ROS). DNA is a major macromolecular target of ROS, and ROS-induced DNA strand breaks activate poly(ADP-ribose)polymerase-1 (PARP-1). Upon activation PARP-1 uses NAD(+) as a substrate to catalyze the transfer of ADP-ribose subunits to a host of nuclear proteins. In the face of extensive DNA strand breaks, PARP-1 activation can lead to depletion of intracellular NAD(P)(H) pools, large decreases in ATP, that threaten cell survival. Accordingly, inhibition of PARP-1 activity after acute oxidative injury has been shown to increase cell survival. When NGF-differentiated PC12 cells, an in vitro neuronal model, are exposed to H(2)O(2) there is increased synthesis of poly ADP-ribose and decreases in intracellular NAD(P)(H) and ATP. Addition of the chemical PARP inhibitor 3-aminobenzamide (AB) prior to H(2)O(2) exposure blocks the synthesis of poly ADP-ribose and maintains intracellular NAD(P)(H) and ATP levels. H(2)O(2) injury is characterized by an immediate, necrotic cell death 2h after injury and a delayed apoptotic-like death 12-24h after injury. This apoptotic-like death is characterized by apoptotic membrane changes and apoptotic DNA fragmentation but is not associated with measurable caspase-3 activity. AB delays cell death beyond 24h and increases cell survival by approximately 25%. This protective effect is accompanied by significantly decreased necrosis and the apoptotic-like death associated with H(2)O(2) exposure. AB also restores caspase-3 which can be attributed to the activation of the upstream activator of caspase-3, caspase-9. Thus, the maintenance of intracellular ATP levels associated with PARP-1 inhibition shifts cell death from necrosis to apoptosis and from apoptosis to cell survival. Furthermore, the shift from necrosis to apoptosis may be explained, in part, by an energy-dependent activation of caspase-9. PMID:15465278

  4. Antimutator Variants of DNA Polymerases

    PubMed Central

    Herr, Alan J.; Williams, Lindsey N.; Preston, Bradley D.

    2011-01-01

    Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino-acid substitutions that lower intrinsic error rates. Here, we review these ‘antimutagenic’ changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino-acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient ‘mutator’ derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino-acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype. PMID:21977975

  5. DNA Polymerase β Ribonucleotide Discrimination

    PubMed Central

    Cavanaugh, Nisha A.; Beard, William A.; Wilson, Samuel H.

    2010-01-01

    DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion. PMID:20519499

  6. Evaluation of the anti-ischemic effects of D-ribose during dobutamine stress echocardiography: a pilot study

    PubMed Central

    Sawada, Stephen G; Lewis, Stephen; Kovacs, Roxanne; Khouri, Samer; Gradus-Pizlo, Irmina; St Cyr, John A; Feigenbaum, Harvey

    2009-01-01

    D-Ribose, a pentose sugar, has shown to improve myocardial high-energy phosphate stores depleted by ischemia. This study investigated the ability of D-Ribose with low dose dobutamine to improve the contractile response of viable myocardium to dobutamine and to assess the efficacy of D-ribose in reducing stress-induced ischemia. Twenty-six patients with ischemic cardiomyopathy completed a two-day, randomized, double blind crossover trial comparing the effects of D-Ribose and placebo on regional wall motion. On the first study day, either D-Ribose or placebo was infused for 4.5 hours. Low (5 and 10 μ/kg/min) and subsequently, high (up to 50 μ/kg/min) dose dobutamine echocardiography was then performed. On the second study day, patients crossed over to the alternative article for a similar 4.5 hours infusion time period and underwent a similar evaluation. The wall motion response during low dose dobutamine was the same with D-Ribose and placebo in 77% of segments (203/263, Kappa = 0.37). In segments with discordant responses, more segments improved with D-Ribose than with placebo (41 vs. 19 segments, p = 0.006). With high dose dobutamine infusion, the wall motion response (ischemia vs. no ischemia) was the same with D-Ribose and placebo in 83% of interpretable segments (301/363, kappa = 0.244). In segments with discordant responses, there were more ischemic segments with placebo compared to D-Ribose (36 vs. 26, p = 0.253). Nineteen patients developed ischemia during the dobutamine and placebo infusion and 13 patients had ischemia during dobutamine and D-ribose infusion (p = 0.109). D-Ribose improved contractile responses to dobutamine in viable myocardium with resting dysfunction but had no significant effect in reducing the frequency of stress-induced wall motion abnormalities. PMID:19200398

  7. Ribose-enhanced myocardial recovery following ischemia in the isolated working rat heart.

    PubMed

    Pasque, M K; Spray, T L; Pellom, G L; Van Trigt, P; Peyton, R B; Currie, W D; Wechsler, A S

    1982-03-01

    Recovery for myocardial adenosine triphosphate (ATP) following moderate periods of ischemic is dependent upon the availability of adenosine monophosphate (AMP) and diphosphate (ADP) for rephosphorylation. Recovery of AMP and ADP levels following ischemia is, in turn, determined by the rates of salvage and de novo adenine nucleotide synthesis. Phosphoribosyl pyrophosphate (PRPP) availability is rate limiting in both salvage and de novo adenine nucleotide synthesis. Parenteral ribose infusions in rats have been documented to elevate myocardial PRPP levels with resultant enhancement of adenine nucleotide synthesis. In this study postischemic recovery of myocardial function and ATP levels in isolated, working rat hearts given ribose infusions before and after ischemia was compared with recovery in control hearts subjected to the same protocol without ribose administration. The mean percent of functional recovery in control hearts following 15 minutes of warm ischemia reached values of 56.7 +/- 4.1%, 63.5% +/- 4.3%, 65.9% +/- 4.6%, and 70.5% +/- 4.7% at 2, 5, 10, and 15 minutes of work following ischemia. Hearts perfused with ribose demonstrated improved mean percent return of function at similar intervals of postischemic work with values of 67.9% +/- 4.2%, 73.7% +/- 3.7%, 81.0% +/- 3.5% (* = p less than 0.02 versus control) *and 85.4% +/- 3.3%, *respectively. Determinations of myocardial ATP levels (mumoles/gm of dry weight) made at the end of 15 minutes of postischemic work were significantly higher (p less than 0.02) in the ribose-treated hearts (18.9 +/- 0.7) than in controls (16.3 +/- 0.6). Infusion of ribose before and after ischemia is a biochemically logical method of improving postischemic myocardial ATP and functional recovery by manipulation of adenine nucleotide synthetic pathways. PMID:6174831

  8. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  9. Trial watch – inhibiting PARP enzymes for anticancer therapy

    PubMed Central

    Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

    2016-01-01

    ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

  10. A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol.

    PubMed

    Sajish, Mathew; Schimmel, Paul

    2015-03-19

    Resveratrol is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects by initiating a stress response that induces survival genes. Because human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) translocates to the nucleus under stress conditions, we considered the possibility that the tyrosine-like phenolic ring of resveratrol might fit into the active site pocket to effect a nuclear role. Here we present a 2.1 Å co-crystal structure of resveratrol bound to the active site of TyrRS. Resveratrol nullifies the catalytic activity and redirects TyrRS to a nuclear function, stimulating NAD(+)-dependent auto-poly-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1). Downstream activation of key stress signalling pathways are causally connected to TyrRS-PARP1-NAD(+) collaboration. This collaboration is also demonstrated in the mouse, and is specifically blocked in vivo by a resveratrol-displacing tyrosyl adenylate analogue. In contrast to functionally diverse tRNA synthetase catalytic nulls created by alternative splicing events that ablate active sites, here a non-spliced TyrRS catalytic null reveals a new PARP1- and NAD(+)-dependent dimension to the physiological mechanism of resveratrol. PMID:25533949

  11. A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

    SciTech Connect

    Yashiroda, Yoko; Hatsugai, Kaori; Takemoto, Yasushi; Saito, Tamio; Sugimoto, Yoshikazu; Osada, Hiroyuki; Yoshida, Minoru

    2010-04-09

    The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

  12. Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

    PubMed

    Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

    2013-06-01

    Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. PMID:23523665

  13. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

    PubMed Central

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L.; Gongol, Brendan; Xiao, Han; Zhang, You-yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  14. Structural insights into SAM domain-mediated tankyrase oligomerization.

    PubMed

    DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

    2016-09-01

    Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. PMID:27328430

  15. PARP INHIBITION ALLEVIATES DIABETES-INDUCED SYSTEMIC OXIDATIVE STRESS AND NEURAL TISSUE 4-HYDROXYNONENAL ADDUCT ACCUMULATION: CORRELATION WITH PERIPHERAL NERVE FUNCTION

    PubMed Central

    Lupachyk, Sergey; Shevalye, Hanna; Maksimchyk, Yury; Drel, Viktor R.; Obrosova, Irina G.

    2011-01-01

    This study evaluated the role of poly(ADP-ribose) polymerase in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg−1d−1, for 10 weeks after initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and non-neuronal cells (fluorescent immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA), 4-hydroxynonenal adduct accumulation in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia (double-label fluorescent immunohistochemistry) as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescent immunohistochemistry) which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy. PMID:21300148

  16. PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression.

    PubMed

    Mariano, Germano; Ricciardi, Maria Rosaria; Trisciuoglio, Daniela; Zampieri, Michele; Ciccarone, Fabio; Guastafierro, Tiziana; Calabrese, Roberta; Valentini, Elisabetta; Tafuri, Agostino; Del Bufalo, Donatella; Caiafa, Paola; Reale, Anna

    2015-06-20

    To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosis. PMID:25938539

  17. PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression

    PubMed Central

    Mariano, Germano; Ricciardi, Maria Rosaria; Trisciuoglio, Daniela; Zampieri, Michele; Ciccarone, Fabio; Guastafierro, Tiziana; Calabrese, Roberta; Valentini, Elisabetta; Tafuri, Agostino; Del Bufalo, Donatella; Caiafa, Paola; Reale, Anna

    2015-01-01

    To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosis. PMID:25938539

  18. [PARP1 inhibitors: contemporary attempts at their use in anticancer therapy and future perspective].

    PubMed

    Wiśnik, Ewelina; Ryksa, Marcin; Koter-Michalak, Maria

    2016-01-01

    Current cancer therapies are based mainly on the use of compounds that cause DNA damage. Unfortunately, even the combination therapies do not give rewarding effects, due to the high efficiency of DNA damage repair mechanisms in tumor cells. Therefore, the present studies should be focused on proteins that are involved in DNA repair systems. Poly(ADP-ribose) polymerase-1 is an example of a protein commonly known as an enzyme that plays a role in the detection of DNA damage and repair. Activation of PARP1 in response to DNA damage leads to poly-ADP-ribosylation of proteins contributing to DNA repair systems, therefore facilitating the maintenance of genome stability. On the other hand, inhibition of PARP1 enzyme results in the accumulation of DNA damage, which in turn contributes to cell death. Studies on inhibitors of PARP1 are still ongoing, and some of them are currently in the third phase of clinical trials. To date, only one representative of the PARP1 inhibitors, called olaparib, has been approved for anti-cancer therapy in the EU and the USA. Moreover, a growing body of evidence indicates a role of this protein in various intracellular processes such as bioenergetics, proliferation, regulation of gene expression, cell death as well as immunoregulation. A number of different intracellular processes regulated by PARP1 give rise to potential wider use of PARP1 inhibitors in treatment of other diseases, including immune or autoimmune disorders. PMID:27117104

  19. Trial watch - inhibiting PARP enzymes for anticancer therapy.

    PubMed

    Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

    2016-03-01

    Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

  20. Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo.

    PubMed

    Yasukawa, Masaaki; Fujihara, Hisako; Fujimori, Hiroaki; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Kishi, Yuta; Hamada, Yoshiki; Masutani, Mitsuko

    2016-01-01

    Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer. PMID:26927065

  1. Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo

    PubMed Central

    Yasukawa, Masaaki; Fujihara, Hisako; Fujimori, Hiroaki; Kawaguchi, Koji; Yamada, Hiroyuki; Nakayama, Ryoko; Yamamoto, Nanami; Kishi, Yuta; Hamada, Yoshiki; Masutani, Mitsuko

    2016-01-01

    Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer. PMID:26927065

  2. [PARP inhibitors and radiotherapy: rational and prospects for a clinical use].

    PubMed

    Pernin, V; Mégnin-Chanet, F; Pennaneach, V; Fourquet, A; Kirova, Y; Hall, J

    2014-12-01

    Poly(ADP-ribosyl)ation is a ubiquitous protein modification involved in the regulation of many cellular processes that is carried out by the poly(ADP-ribose) polymerase (PARP) family. The PARP-1, PARP-2 and PARP-3 are the only PARPs known to be activated by DNA damage. The absence of PARP-1 and PARP-2, that are both activated by DNA damage and participate in DNA damage repair processes, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD(+) at the enzyme's activity site can be used in BRCA-deficient cells as single agent therapies acting through the principle of synthetic lethality exploiting these cells deficient DNA double-strand break repair. Preclinical data showing an enhancement of the response of tumors to radiation has been documented for several PARP inhibitors. However, whether this is due exclusively to impaired DNA damage responses or whether tumor re-oxygenation contributes to this radio-sensitization via the vasoactive effects of the PARP inhibitors remains to be fully determined. These promising results have paved the way for the evaluation of PARP inhibitors in combination with radiotherapy in phase I and phase II clinical trials for malignant glioma, head and neck, and breast cancers. A number of challenges remain that are also reviewed in this article, including the optimization of treatment schedules for combined therapies and the validation of biomarkers that will identify which patients will most benefit from either PARP inhibitors in combination with radiotherapy. PMID:25441760

  3. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade.

    PubMed

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L; Gongol, Brendan; Xiao, Han; Zhang, You-Yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  4. Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines

    PubMed Central

    Ávila-Arroyo, Sonia; Nuñez, Gema Santamaría; García-Fernández, Luis Francisco

    2015-01-01

    Purpose Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells. Methods We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively. Results The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib. Conclusion Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status. PMID:26770239

  5. PARP-1 transcriptional activity is regulated by sumoylation upon heat shock.

    PubMed

    Martin, Nadine; Schwamborn, Klaus; Schreiber, Valérie; Werner, Andreas; Guillier, Christelle; Zhang, Xiang-Dong; Bischof, Oliver; Seeler, Jacob-S; Dejean, Anne

    2009-11-18

    Heat shock and other environmental stresses rapidly induce transcriptional responses subject to regulation by a variety of post-translational modifications. Among these, poly(ADP-ribosyl)ation and sumoylation have received growing attention. Here we show that the SUMO E3 ligase PIASy interacts with the poly(ADP-ribose) polymerase PARP-1, and that PIASy mediates heat shock-induced poly-sumoylation of PARP-1. Furthermore, PIASy, and hence sumoylation, appears indispensable for full activation of the inducible HSP70.1 gene. Chromatin immunoprecipitation experiments show that PIASy, SUMO and the SUMO-conjugating enzyme Ubc9 are rapidly recruited to the HSP70.1 promoter upon heat shock, and that they are subsequently released with kinetics similar to PARP-1. Finally, we provide evidence that the SUMO-targeted ubiquitin ligase RNF4 mediates heat-shock-inducible ubiquitination of PARP-1, regulates the stability of PARP-1, and, like PIASy, is a positive regulator of HSP70.1 gene activity. These results, thus, point to a novel mechanism for regulating PARP-1 transcription function, and suggest crosstalk between sumoylation and RNF4-mediated ubiquitination in regulating gene expression in response to heat shock. PMID:19779455

  6. Data on four apoptosis-related genes in the colonial tunicate Botryllus schlosseri.

    PubMed

    Franchi, Nicola; Ballin, Francesca; Manni, Lucia; Schiavon, Filippo; Ballarin, Loriano

    2016-09-01

    The data described are related to the article entitled "Recurrent phagocytosis-induced apoptosis in the cyclical generation change of the compound ascidian Botryllus schlosseri" (Franchi et al., 2016) [1]. Four apoptosis-related genes, showing high similarity with mammalian Bax (a member of the Bcl-2 protein family), AIF1 (apoptosis-inducing factor-1), PARP1 (poly ADP ribose polymerase-1) and IAP7 (inhibitor of apoptosis-7) were identified from the analysis of the trascriptome of B. schlosseri. They were named BsBax, BsAIF1, BsPARP1 and BsIAP7. Here, their deduced amino acid sequence were compared with known sequences of orthologous genes from other deuterostome species together with a study of their identity/similarity. PMID:27294183

  7. Convallatoxin, a Dual Inducer of Autophagy and Apoptosis, Inhibits Angiogenesis In Vitro and In Vivo

    PubMed Central

    Yang, Seung Ya; Kim, Nam Hee; Cho, Yoon Sun; Lee, Hukeun; Kwon, Ho Jeong

    2014-01-01

    Autophagy and apoptosis are important processes that control cellular homeostasis and have been highlighted as promising targets for novel cancer therapies. Here, we identified convallatoxin (CNT), isolated from Antiaris toxicaria, as a dual inducer of autophagy and apoptosis. CNT exerts cytotoxic effects on a number of cancer and normal cell lines and induces apoptosis by increasing caspase-3 and poly ADP ribose polymerase (PARP) cleavage. Moreover, dose- and time-dependent autophagic activity was detected in CNT-treated cells, and mammalian target of rapamycin (mTOR)/p70S6K signal pathway inhibition was observed. Notably, CNT inhibits human umbilical vein endothelial cell (HUVEC) growth and exerts anti-angiogenic activity in vitro and in vivo. Collectively, these results demonstrate that the naturally occurring compound, CNT, is a novel anti-angiogenic compound via dual inducing of autophagy and apoptosis. PMID:24663328

  8. Recurrent Glioblastoma: Where we stand

    PubMed Central

    Roy, Sanjoy; Lahiri, Debarshi; Maji, Tapas; Biswas, Jaydip

    2015-01-01

    Current first-line treatment regimens combine surgical resection and chemoradiation for Glioblastoma that provides a slight increase in overall survival. Age on its own should not be used as an exclusion criterion of glioblastoma multiforme (GBM) treatment, but performance should be factored heavily into the decision-making process for treatment planning. Despite aggressive initial treatment, most patients develop recurrent diseases which can be treated with re-resection, systemic treatment with targeted agents or cytotoxic chemotherapy, reirradiation, or radiosurgery. Research into novel therapies is investigating alternative temozolomide regimens, convection-enhanced delivery, immunotherapy, gene therapy, antiangiogenic agents, poly ADP ribose polymerase inhibitors, or cancer stem cell signaling pathways. Given the aggressive and resilient nature of GBM, continued efforts to better understand GBM pathophysiology are required to discover novel targets for future therapy. PMID:26981507

  9. PARP activation promotes nuclear AID accumulation in lymphoma cells

    PubMed Central

    Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-01-01

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma. PMID:26921193

  10. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells

    PubMed Central

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  11. The caspase 3-dependent apoptotic effect of pycnogenol in human oral squamous cell carcinoma HSC-3 cells.

    PubMed

    Yang, In-Hyoung; Shin, Ji-Ae; Kim, Lee-Han; Kwon, Ki Han; Cho, Sung-Dae

    2016-01-01

    In the present study, the apoptotic effect of pycnogenol and its molecular mechanism in human oral squamous cell carcinoma HSC-3 cells were investigated. Pycnogenol significantly inhibited the viability of HSC-3 cells and suppressed neoplastic cell transformation in HSC-3 cells and TPA-treated JB6 cells. It caused caspase-dependent apoptosis evidenced by the increase in cleaved poly (ADP-ribose) polymerase and caspase 3 in a dose-dependent manner. Pycnogenol increased Bak protein by enhancing its protein stability whereas other Bcl-2 family members were not altered. In addition, the treatment with pycnogenol led to the production of reactive oxygen species and N-acetyl-l-cysteine almost blocked pycnogenol-induced reactive oxygen species generation. Taken together, these findings suggest that pycnogenol may be a potential candidate for the chemoprevention or chemotherapy of human oral cancer. PMID:26798196

  12. Profile of veliparib and its potential in the treatment of solid tumors

    PubMed Central

    Wagner, Lars M

    2015-01-01

    Inhibition of poly(ADP-ribose) polymerase (PARP) is an attractive therapeutic strategy because of the importance of this pathway in restoring DNA damage. Small-molecule inhibitors of PARP appear most effective when used to treat tumors with underlying defects in DNA repair, or when combined with DNA-damaging agents. Veliparib is one of several recently developed oral inhibitors of PARP currently in clinical trials. This review summarizes the pharmacology, mechanisms of action, toxicity, and activity of veliparib seen in clinical trials to date. Also discussed are proposed mechanisms of resistance, potential biomarkers of activity, and issues regarding patient selection and combination therapies that may optimize use of this exciting new agent. PMID:26251615

  13. Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death.

    PubMed

    Wang, Hongmin; Yu, Seong-Woon; Koh, David W; Lew, Jasmine; Coombs, Carmen; Bowers, William; Federoff, Howard J; Poirier, Guy G; Dawson, Ted M; Dawson, Valina L

    2004-12-01

    The profound neuroprotection observed in poly(ADP-ribose) polymerase-1 (PARP-1) null mice to ischemic and excitotoxic injury positions PARP-1 as a major mediator of neuronal cell death. We report here that apoptosis-inducing factor (AIF) mediates PARP-1-dependent glutamate excitotoxicity in a caspase-independent manner after translocation from the mitochondria to the nucleus. In primary murine cortical cultures, neurotoxic NMDA exposure triggers AIF translocation, mitochondrial membrane depolarization, and phosphatidyl serine exposure on the cell surface, which precedes cytochrome c release and caspase activation. NMDA neurotoxicity is not affected by broad-spectrum caspase inhibitors, but it is prevented by Bcl-2 overexpression and a neutralizing antibody to AIF. These results link PARP-1 activation with AIF translocation in NMDA-triggered excitotoxic neuronal death and provide a paradigm in which AIF can substitute for caspase executioners. PMID:15574746

  14. Recurrent Glioblastoma: Where we stand.

    PubMed

    Roy, Sanjoy; Lahiri, Debarshi; Maji, Tapas; Biswas, Jaydip

    2015-01-01

    Current first-line treatment regimens combine surgical resection and chemoradiation for Glioblastoma that provides a slight increase in overall survival. Age on its own should not be used as an exclusion criterion of glioblastoma multiforme (GBM) treatment, but performance should be factored heavily into the decision-making process for treatment planning. Despite aggressive initial treatment, most patients develop recurrent diseases which can be treated with re-resection, systemic treatment with targeted agents or cytotoxic chemotherapy, reirradiation, or radiosurgery. Research into novel therapies is investigating alternative temozolomide regimens, convection-enhanced delivery, immunotherapy, gene therapy, antiangiogenic agents, poly ADP ribose polymerase inhibitors, or cancer stem cell signaling pathways. Given the aggressive and resilient nature of GBM, continued efforts to better understand GBM pathophysiology are required to discover novel targets for future therapy. PMID:26981507

  15. Lantana camara Induces Apoptosis by Bcl-2 Family and Caspases Activation.

    PubMed

    Han, Eun Byeol; Chang, Bo Yoon; Jung, Young Suk; Kim, Sung Yeon

    2015-04-01

    Breast cancer is one of the most common cancers worldwide, and the second most fatal cancer in women after lung cancer. Because there are instances of cancer resistance to existing therapies, studies focused on the identification of novel therapeutic drugs are very important. In this study, we identified a natural anticancer agent from Lantana camara, a flowering plant species of the genus Verbena. The extract obtained from the L. camara exhibited cell death properties in the human breast cancer cell line, MCF-7. We found that the apoptosis induced by treatment with the L. camara extract was regulated by the Bcl-2 family. Bid and Bax was increased and Bcl-2 was decreased by L. camara extract. L. camara extract modulated cleavage of caspase-8, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP). Our results support the potential use of the L. camara extract as an anti-breast cancer drug. PMID:25145450

  16. New treatment options for advanced pancreatic cancer.

    PubMed

    Middleton, Gary; Ghaneh, Paula; Costello, Eithne; Greenhalf, William; Neoptolemos, John P

    2008-10-01

    Pancreatic cancer has a very high mortality rate and affects approximately 230,000 individuals worldwide. Gemcitabine has become established as the standard therapy for advanced pancreatic cancer; however, the survival advantage is small. Adjuvant chemotherapy using either 5-fluorouracil or gemcitabine is now established in pancreatic cancer as an alternative therapy. Combinations of gemcitabine with either platin agents or capecitabine may be advantageous. Anti-EGFR and anti-VEGF agents have been unsuccessful but multiple tyrosine kinase inhibitors are under investigation. Of the increasing number of immunological agents, the GV1001 antitelomerase vaccine holds some interest. Targeted agents against important mitogenic pathways, including MEK/ERK, Src, PI3K/Akt, mTOR, Hedgehog and NF-kappaB, as well as agents targeting histone deacetylase, poly(ADP-ribose) polymerase, heat shock protein 90 and other agents such as beta-lapachone, hold considerable interest for further development. However, the probability of individual success is low. PMID:19072345

  17. Consumer beware: a systematic assessment of potential bias in the lay electronic media to examine the portrayal of "PARP" inhibitors for cancer treatment.

    PubMed

    Coleman, Shawnta; Peethambaram, Prema P; Jatoi, Aminah

    2011-09-01

    This study examined how the lay electronic media covers poly-ADP-ribose polymerase, or "PARP," inhibitors, a class of cancer agents currently under clinical investigation. Of 771 internet links, 51 targeted the lay public. Independent review by two investigators yielded the following categorizations: 36 (71%) were "overly positive", 15 (29%) "neutral", and none "overly negative". "Overly positive" articles used: (l) overstated benefit, (2) included quotations from enthusiastic scientists, and (3) discussed single or small patient subsets. They used such phrases as "the holy grail of cancer research", "the most exciting development in cancer research in a decade or more…. it could save thousands of lives", and "we were surprised and delighted…. it's the kind of thing you don't really think will happen". Healthcare providers should be aware of the foregoing when discussing PARP inhibitors-and perhaps other novel therapies-with cancer patients. PMID:20857352

  18. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

    PubMed Central

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V.; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy. PMID:24682826

  19. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    2005-12-30

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.

  20. Escin sodium induces apoptosis of human acute leukemia Jurkat T cells.

    PubMed

    Zhang, Zhenzhen; Gao, Jian; Cai, Xueting; Zhao, Youlong; Wang, Yafei; Lu, Wuguang; Gu, Zhenhua; Zhang, Shuangquan; Cao, Peng

    2011-12-01

    Escin sodium has been used in the clinic as an antioedematous, antiexudative and vasoprotective agent for many years and has shown excellent tolerability. However, little is known about its anticancer activity. This is a report for the first time that escin sodium exerts a cytotoxic effect on human acute leukemia Jurkat T cells via the induction of apoptosis rather than cell cycle arrest. Escin sodium activated the initiator caspase-8, -9, and the effector caspase-3, degraded poly (ADP-ribose) polymerase (PARP) and attenuated the expression of Bcl-2. In addition, escin sodium inhibited the growth of cancer cells in a selective manner with Jurkat cells most sensitive to it. Taken together, the data show that escin sodium possesses potent apoptogenic activity toward human acute leukemia Jurkat T cells. PMID:21452372

  1. Repurposing an Old Drug to Improve the Use and Safety of Tissue Plasminogen Activator for Acute Ischemic Stroke: Minocycline

    PubMed Central

    Hess, David C.; Fagan, Susan C.

    2015-01-01

    There is only 1 US Food and Drug Administration–approved drug for acute ischemic stroke: tissue plasminogen activator (tPA). Due to a short time window and fear of intracerebral hemorrhage (ICH), tPA remains underutilized. There is great interest in developing combination drugs to use with tPA to improve the odds of a favorable recovery and to reduce the risk of ICH. Minocycline is a broad-spectrum antibiotic that has been found to be a neuroprotective agent in preclinical ischemic stroke models. Minocycline inhibits matrix metalloproteinase-9, a biomarker for ICH associated with tPA use. Minocycline is also an anti-inflammatory agent and inhibits poly (ADP-ribose) polymerase-1. Minocycline has been safe and well tolerated in the clinical trials conducted to date. PMID:20410869

  2. Selective inhibition of PARP10 using a chemical genetics strategy.

    PubMed

    Morgan, Rory K; Carter-O'Connell, Ian; Cohen, Michael S

    2015-11-01

    The lack of inhibitors that are selective for individual poly-ADP-ribose polymerase (PARP) family members has limited our understanding of their roles in cells. Here, we describe a chemical genetics approach for generating selective inhibitors of an engineered variant of PARP10. We synthesized a series of C-7 substituted 3,4-dihydroisoquinolin-1(2H)-one (dq) analogues designed to selectively inhibit a mutant of PARP10 (LG-PARP10) that contains a unique pocket in its active site. A dq analogue containing a bromo at the C-7 position demonstrated a 10-fold selectivity for LG-PARP10 compared to its WT counterpart. This study provides a platform for the development of selective inhibitors of individual PARP family members that will be useful for decoding their cellular functions. PMID:26231158

  3. p53 regulates a non-apoptotic death induced by ROS

    PubMed Central

    Montero, J; Dutta, C; van Bodegom, D; Weinstock, D; Letai, A

    2013-01-01

    DNA damage induced by reactive oxygen species and several chemotherapeutic agents promotes both p53 and poly (ADP-ribose) polymerase (PARP) activation. p53 activation is well known to regulate apoptotic cell death, whereas robust activation of PARP-1 has been shown to promote a necrotic cell death associated with energetic collapse. Here we identify a novel role for p53 in modulating PARP enzymatic activity to regulate necrotic cell death. In mouse embryonic fibroblasts, human colorectal and human breast cancer cell lines, loss of p53 function promotes resistance to necrotic, PARP-mediated cell death. We therefore demonstrate that p53 can regulate both necrotic and apoptotic cell death, mutations or deletions in this tumor-suppressor protein may be selected by cancer cells to provide not only their resistance to apoptosis but also to necrosis, and explain resistance to chemotherapy and radiation even when it kills via non-apoptotic mechanisms. PMID:23703322

  4. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    PubMed

    Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

    2014-06-01

    Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

  5. NAD+ and sirtuins in aging and disease.

    PubMed

    Imai, Shin-ichiro; Guarente, Leonard

    2014-08-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide. PMID:24786309

  6. The Sarin-like Organophosphorus Agent bis (isopropyl methyl)phosphonate Induces Apoptotic Cell Death and COX-2 Expression in SK-N-SH Cells.

    PubMed

    Arima, Yosuke; Yoshimoto, Kanji; Namera, Akira; Makita, Ryosuke; Murata, Kazuhiro; Nagao, Masataka

    2016-03-01

    Organophosphorus compounds, such as sarin, are highly toxic nerve agents that inhibit acetylcholinesterase (AChE), but not cholinesterase, via multiple mechanisms. Recent studies have shown that organophosphorus compounds increase cyclooxygenase-2 (COX-2) expression and induce neurotoxicity. In this study, we examined the toxicity of the sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate (BIMP) and the effects of BIMP on COX-2 expression in SK-N-SH human neuroblastoma cells. Exposure to BIMP changed cell morphology and induced caspase-dependent apoptotic cell death accompanied by cleavage of caspase 3, caspase 9, and poly (ADP-ribose) polymerase (PARP). It also increased COX-2 expression, while pretreatment with a COX inhibitor, ibuprofen, decreased BIMP-dependent cell death and COX-2 expression in SK-N-SH cells. Thus, our findings suggest that BIMP induces apoptotic cell death and upregulates COX-2 expression. PMID:27348899

  7. Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease

    PubMed Central

    Lehmann, S; Costa, A C; Celardo, I; Loh, S H Y; Martins, L M

    2016-01-01

    The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction. PMID:27031963

  8. Novel targeted therapeutics for mantle cell lymphoma--what's on the horizon?

    PubMed

    El Halabi, Layal; Ghez, David; Ribrag, Vincent

    2016-03-01

    Major advances have significantly improved the outcome of mantle cell lymphoma (MCL). Incorporation of rituximab to CHOP regimen, the adoption of high dose cytarabine with frontline autologous stem cell transplantation in young patients, maintenance rituximab or bortezomib based chemotherapy in elderly patients, improved the disease outcome. Bortezomib, lenalidomide, temsirolimus and ibrutinib have proven their efficacy and are approved for the use in refractory or relapsed MCL patients. Several other molecules are currently being evaluated such as cyclin dependent kinase 4/6 (CDK4/6), phosphoinositide 3-kinase (PI3K), B cell lymphoma-2 (BCL2) and Poly ADP-ribose polymerase (PARP) inhibitors. Unfortunately, we don't have specific biomarkers that could reveal which of the underlying pathways or genetic alterations are mostly involved in each individual case of MCL. Efforts should be done in this field aiming to an optimal personalized therapy. PMID:26689790

  9. Design, Synthesis, and Biological Evaluation of Novel PARP-1 Inhibitors Based on a 1H-Thieno[3,4-d] Imidazole-4-Carboxamide Scaffold.

    PubMed

    Wang, Lingxiao; Liu, Feng; Jiang, Ning; Zhou, Wenxia; Zhou, Xinbo; Zheng, Zhibing

    2016-01-01

    A series of poly(ADP-ribose)polymerase (PARP)-1 inhibitors containing a novel scaffold, the 1H-thieno[3,4-d]imidazole-4-carboxamide moiety, was designed and synthesized. These efforts provided some compounds with relatively good PARP-1 inhibitory activity, and among them, 16l was the most potent one. Cellular evaluations indicated that the anti-proliferative activities of 16g, 16i, 16j and 16l against BRCA-deficient cell lines were similar to that of olaparib, while the cytotoxicities of 16j and 16l toward human normal cells were lower. In addition, ADMET prediction results indicated that these compounds might possess more favorable toxicity and pharmacokinetic properties. This study provides a basis for our further investigation. PMID:27304949

  10. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    PubMed

    Nagy, Zita; Kalousi, Alkmini; Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-02-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  11. Apoptosis induced by the Tibetan herbal remedy PADMA 28 in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2

    PubMed Central

    Jenny, Marcel; Schwaiger, Wolfgang; Bernhard, David; Wrulich, Oliver A; Cosaceanu, Daria; Fuchs, Dietmar; Ueberall, Florian

    2005-01-01

    The Tibetan herbal remedy PADMA 28 revealed promising results to support treatment of atherosclerosis, Charot syndrome (intermittent claudication), chronic active hepatitis and infection of the respiratory tract. The remedy was confirmed to be closely linked with anti- and pro-oxidative properties in vitro. In this study, apoptogenic and survival effects of PADMA 28 were investigated in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2. PADMA 28 led to a concentration-dependent inhibition of cell proliferation accompanied by the accumulation of CEM-C7H2 cells in subG1 phase, fragmentation of poly (ADP-ribose) polymerase (PARP) and nuclear body formation. Treatment with PADMA 28 rescued to some extent cells over-expressing Bcl-2 from apoptosis. This finding suggests that the mechanism of action of PADMA 28 may be via interference with Bcl-2 triggered survival pathways. PMID:16138918

  12. Development of novel dual binders as potent, selective, and orally bioavailable tankyrase inhibitors.

    PubMed

    Hua, Zihao; Bregman, Howard; Buchanan, John L; Chakka, Nagasree; Guzman-Perez, Angel; Gunaydin, Hakan; Huang, Xin; Gu, Yan; Berry, Virginia; Liu, Jingzhou; Teffera, Yohannes; Huang, Liyue; Egge, Bryan; Emkey, Renee; Mullady, Erin L; Schneider, Steve; Andrews, Paul S; Acquaviva, Lisa; Dovey, Jennifer; Mishra, Ankita; Newcomb, John; Saffran, Douglas; Serafino, Randy; Strathdee, Craig A; Turci, Susan M; Stanton, Mary; Wilson, Cindy; Dimauro, Erin F

    2013-12-27

    Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting β-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies. PMID:24294969

  13. Pharmacotherapy for recurrent ovarian cancer: current status and future perspectives.

    PubMed

    Matsumoto, Koji; Onda, Takashi; Yaegashi, Nobuo

    2015-05-01

    Several 'lines of therapy' that utilize cytotoxic agents and are driven by platinum-free intervals are the current standard of care for patients with recurrent ovarian cancer. For patients with platinum-resistant disease, single agent chemotherapy (pegylated liposomal doxorubicin, topotecan, gemcitabine or weekly paclitaxel) is the standard of care. For patients with platinum-sensitive disease, combination chemotherapy (carboplatin plus paclitaxel, pegylated liposomal doxorubicin or gemcitabine) is the standard of care. In addition, antiangiogenic therapy using bevacizumab is an established option. Future directions could include 'lines of therapy' with biologic agents driven by specific biologic targets. Data from antiangiogenic agents (trebananib, pazopanib and cediranib), antifolate drugs (farletuzumab and vintafolide), poly(ADP-ribose) polymerase inhibitors (olaparib and veliparib), mTOR inhibitors (everolimus and temsirolimus) and immune editing agents (nivolumab) have been summarized in this review. PMID:25765457

  14. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

    PubMed Central

    Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-01-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  15. Structural studies of large nucleoprotein particles, vaults.

    PubMed

    Tanaka, Hideaki; Tsukihara, Tomitake

    2012-01-01

    Vault is the largest nonicosahedral cytosolic nucleoprotein particle ever described. The widespread presence and evolutionary conservation of vaults suggest important biologic roles, although their functions have not been fully elucidated. X-ray structure of vault from rat liver was determined at 3.5 Å resolution. It exhibits an ovoid shape with a size of 40 × 40 × 67 nm(3). The cage structure of vault consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The other components of vaults, telomerase-associated proteins, poly(ADP-ribose) polymerases and small RNAs, are in location in the vault particle by electron microscopy. PMID:23060231

  16. Vaults and the major vault protein: novel roles in signal pathway regulation and immunity.

    PubMed

    Berger, W; Steiner, E; Grusch, M; Elbling, L; Micksche, M

    2009-01-01

    The unique and evolutionary highly conserved major vault protein (MVP) is the main component of ubiquitous, large cellular ribonucleoparticles termed vaults. The 100 kDa MVP represents more than 70% of the vault mass which contains two additional proteins, the vault poly (ADP-ribose) polymerase (vPARP) and the telomerase-associated protein 1 (TEP1), as well as several short untranslated RNAs (vRNA). Vaults are almost ubiquitously expressed and, besides chemotherapy resistance, have been implicated in the regulation of several cellular processes including transport mechanisms, signal transmissions and immune responses. Despite a growing amount of data from diverse species and systems, the definition of precise vault functions is still highly complex and challenging. Here we review the current knowledge on MVP and vaults with focus on regulatory functions in intracellular signal transduction and immune defence. PMID:18759128

  17. Dieckol, isolated from Ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma Hep3B cells.

    PubMed

    Yoon, Jin-Soo; Kasin Yadunandam, Anandam; Kim, Soon-Jin; Woo, Hee-Chul; Kim, Hyeung-Rak; Kim, Gun-Do

    2013-07-01

    Phlorotannins have been reported to demonstrate several biological properties, including antioxidant activity, and activities useful in the treatment of diabetic complications and in chemoprevention of several vascular diseases. In this study, we focused on the apoptosis induced by dieckol, a marine algal phlorotannin isolated from Ecklonia stolonifera, on human hepatocellular carcinoma (HCC) Hep3B cells. Dieckol reduced the numbers of viable cells and increased the numbers of apoptotic cells in a dose-dependent manner. Immunoblotting analysis revealed that dieckol increased the expression levels of cleaved caspases-3, 7, 8, and 9, and cleaved poly(ADP-ribose) polymerase. Dieckol increased the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with apoptosis-inducing factor. In addition, dieckol induced increased expression of truncated Bid and Bim. The results indicate that dieckol induces apoptosis via the activation of both death receptor and mitochondrial-dependent pathways in HCC Hep3B cells. PMID:23054486

  18. Prevention of cellular oxidative damage by an aqueous extract of Anoectochilus formosanus.

    PubMed

    Wang, Leng-Fang; Lin, Chun-Mao; Shih, Chwen-Ming; Chen, Hui-Ju; Su, Borcherng; Tseng, Cheng-Chuang; Gau, Bao-Bih; Cheng, Kur-Ta

    2005-05-01

    Anoectochilus formosanus (AF) is a popular folk medicine in Taiwan whose pharmacological effects have been characterized. In this work we investigated the antioxidant properties of an aqueous extract prepared from AF. The AF extract was capable of scavenging H2O2 in a dose-dependent manner. We induced oxidative stress in HL-60 cells, either by the addition of hydrogen peroxide (H2O2) or by the xanthine/xanthine oxidase reaction. Apoptosis caused by oxidative damage was displayed by DNA fragmentation on gel electrophoresis, and the apoptotic fraction was quantified with flow cytometry. The cell damage induced by oxidative stress was prevented by the plant extract in a concentration-dependent manner. Furthermore, the proteolytic cleavage of poly(ADP-ribose) polymerase during the apoptotic process was also inhibited by AF extract. Our results provide the basis for determining an AF extract to be an antioxidant. PMID:15965084

  19. Niacinamide - mechanisms of action and its topical use in dermatology.

    PubMed

    Wohlrab, Johannes; Kreft, Daniela

    2014-01-01

    Niacinamide, an amide of vitamin B3 (niacin), is a hydrophilic endogenous substance. Its effects after epicutaneous application have long been described in the literature. Given a sufficient bioavailability, niacinamide has antipruritic, antimicrobial, vasoactive, photo-protective, sebostatic and lightening effects depending on its concentration. Within a complex metabolic system niacinamide controls the NFκB-mediated transcription of signalling molecules by inhibiting the nuclear poly (ADP-ribose) polymerase-1 (PARP-1). Niacinamide is a well-tolerated and safe substance often used in cosmetics. Clinical data for its therapeutic use in various dermatoses can increasingly be found in the literature. Although the existing data are not sufficient for a scientifically founded evaluation, it can be stated that the use of niacinamide in galenic preparations for epicutaneous application offers most interesting prospects. PMID:24993939

  20. Identification of novel PARP-1 inhibitors by structure-based virtual screening.

    PubMed

    Hannigan, Kevin; Kulkarni, Shridhar S; Bdzhola, Volodymyr G; Golub, Andriy G; Yarmoluk, Sergiy M; Talele, Tanaji T

    2013-11-01

    Poly(ADP-ribose)polymerase-1 (PARP-1) is an abundant and ubiquitous chromatin-bound nuclear protein. PARP-1, a DNA repair enzyme, has been in the limelight as a chemotherapeutic target. In this study, we demonstrated the successful use of structure-based virtual screening to identify inhibitors of PARP-1 from Otava databases comprised of nearly 260,000 compounds. Five novel inhibitors belonging to thienopyrimidinone, isoquinolinoquinazolinone, pyrroloquinazolinone, and cyclopentenothienopyrimidinone scaffolds revealed inhibitory potencies with IC50 values ranged from 9.57μM to 0.72μM. Structural features relevant to the activity of these novel compounds within the active site of PARP-1 are discussed in detail and will guide future SAR investigation on these scaffolds. PMID:24074844

  1. Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells.

    PubMed

    Ramamoorthy, Mahesh; Smith, Susan

    2015-09-14

    The chromatin-remodeler ATRX is frequently lost in cancer cells that use ALT (alternative lengthening of telomeres) for telomere maintenance, but its function in telomere recombination is unknown. Here we show that loss of ATRX suppresses the timely resolution of sister telomere cohesion that normally occurs prior to mitosis. In the absence of ATRX, the histone variant macroH2A1.1 binds to the poly(ADP-ribose) polymerase tankyrase 1, preventing it from localizing to telomeres and resolving cohesion. The resulting persistent telomere cohesion promotes recombination between sister telomeres, while it suppresses inappropriate recombination between non-sisters. Forced resolution of sister telomere cohesion induces excessive recombination between non-homologs, genomic instability, and impaired cell growth, indicating the ATRX-macroH2A1.1-tankyrase axis as a potential therapeutic target in ALT tumors. PMID:26373281

  2. Anti-lipid peroxidation and induction of apoptosis in the erythroleukaemic cell line K562 by extracts from (Tunisian) Rhamnus alaternus L. (Rhamnaceae).

    PubMed

    Ammar, Rebai Ben; Neffati, Aicha; Skandrani, Ines; Sghaier, Mohamed Ben; Bhouri, Wissem; Ghedira, Kamel; Chekir-Ghedira, Leila

    2011-07-01

    Total oligomer flavonoids (TOF) enriched and ethyl acetate (EA) extracts from Rhamnus alaternus induce apoptotic death in human chronic myelogenous leukaemia K562 cell line, as demonstrated by gel electrophoresis, which demonstrates the characteristic ladder patterns of DNA fragmentation and the proteolytic cleavage of poly(ADP ribose) polymerase (PARP). The effect of R. alaternus extract in reducing oxidative stress was evaluated by anti-lipid peroxidation which was monitored by measuring malondialdehyde level in K562 cultured cells. The TOF and EA extracts were found to be effective to protect against lipid peroxidation. Their IC₅₀ values were 196 and 273 µg mL⁻¹, respectively. These findings suggest that R. alaternus extracts exhibit potential antioxidant and proapoptotic properties. PMID:21726127

  3. Synthesis and in vitro Evaluation of 3H-Pyrrolo[3,2-f]-quinolin-9-one Derivatives That Show Potent and Selective Anti-leukemic Activity

    PubMed Central

    Bortolozzi, Roberta; Brun, Paola; Castagliuolo, Ignazio; Hamel, Ernest; Basso, Giuseppe; Viola, Giampietro

    2010-01-01

    A series of new substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-9-ones were synthesized and evaluated for their antiproliferative activity. The most active derivatives showed high selectivity against human leukemia cell lines and potently inhibited their growth, with GI50 values in the nanomolar range. The active compounds strongly blocked tubulin assembly and colchicine binding to tubulin. Their activities were equal to or greater than that of the reference compound combretastatin A-4. Flow cytometry studies showed that the two most active compounds arrested Jurkat cells in the G2/M cell-cycle phase in a concentration-dependent manner. This effect was associated with apoptosis, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and cleavage of the enzyme poly(ADP-ribose) polymerase. PMID:20629070

  4. BRCA somatic mutations and epigenetic BRCA modifications in serous ovarian cancer.

    PubMed

    Moschetta, M; George, A; Kaye, S B; Banerjee, S

    2016-08-01

    The significant activity of poly(ADP-ribose)polymerase (PARP) inhibitors in the treatment of germline BRCA mutation-associated ovarian cancer, which represents ∼15% of HGS cases, has recently led to European Medicines Agency and food and drug administration approval of olaparib. Accumulating evidence suggests that PARP inhibitors may have a wider application in the treatment of sporadic ovarian cancers. Up to 50% of HGS ovarian cancer patients may exhibit homologous recombination deficiency (HRD) through mechanisms including germline BRCA mutations, somatic BRCA mutations, and BRCA promoter methylation. In this review, we discuss the role of somatic BRCA mutations and BRCA methylation in ovarian cancer. There is accumulating evidence for routine somatic BRCA mutation testing, but the relevance of BRCA epigenetic modifications is less clear. We explore the challenges that need to be addressed if the full potential of these markers of HRD is to be utilised in clinical practice. PMID:27037296

  5. Synovial sarcoma cell lines showed reduced DNA repair activity and sensitivity to a PARP inhibitor.

    PubMed

    Yamasaki, Hiroyuki; Miyamoto, Mamiko; Yamamoto, Yuki; Kondo, Tadashi; Watanabe, Toshiki; Ohta, Tsutomu

    2016-08-01

    Synovial sarcoma is a soft-tissue sarcoma and a rare type of cancer. Unfortunately, effective chemotherapies for synovial sarcomas have not been established. In this report, we show that synovial sarcoma cell lines have reduced repair activity for DNA damage induced by ionizing radiation (IR) and a topoisomerase II inhibitor (etoposide). We also observed reduced recruitment of RAD51 homologue (S. cerevisiae; RAD51) at sites of double-strand breaks (DSBs) in synovial sarcoma cell lines that had been exposed to IR. These findings showed that synovial sarcoma cell lines are defective in homologous recombination (HR) repair. Furthermore, we found that a poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281; olaparib) effectively reduced the growth of synovial sarcoma cell lines in the presence of an alkylating agent (temozolomide). Our findings offer evidence that treatment combining a PARP inhibitor and an alkylating agent could have therapeutic benefits in the treatment of synovial sarcoma. PMID:27353471

  6. What Is the Place of PARP Inhibitors in Ovarian Cancer Treatment?

    PubMed

    Liu, Joyce F; Matulonis, Ursula A

    2016-05-01

    Poly-ADP-ribose polymerase (PARP) inhibitors have been one of the most exciting developments in the treatment of ovarian cancer in recent years. Demonstration of anti-cancer activity has led to the European Medicines Agency (EMA) approval of the PARP inhibitor (PARPi) olaparib as maintenance therapy in women with BRCA-mutated (BRCAm) ovarian cancer with platinum-sensitive recurrence following response to platinum therapy and the US Food and Drug Administration (US FDA) approval of olaparib in relapsed germline BRCA-mutated (gBRCAm) ovarian cancer in women who have received at least three prior chemotherapy treatments, both occurring in 2014. Additional trials are underway or awaiting final analysis with olaparib, other PARPis, and PARPi combinations to further elucidate the activity of these drugs in various clinical settings. This review will focus on the current clinical experience and ongoing trials with PARPis in ovarian cancer. PMID:26984416

  7. In vivo imaging of specific drug-target binding at subcellular resolution

    NASA Astrophysics Data System (ADS)

    Dubach, J. M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L. A.; Weissleder, R.

    2014-05-01

    The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.

  8. Characterization of TP53 and PI3K signaling pathways as molecular targets in gynecologic malignancies.

    PubMed

    Oda, Katsutoshi; Ikeda, Yuji; Kashiyama, Tomoko; Miyasaka, Aki; Inaba, Kanako; Fukuda, Tomohiko; Asada, Kayo; Sone, Kenbun; Wada-Hiraike, Osamu; Kawana, Kei; Osuga, Yutaka; Fujii, Tomoyuki

    2016-07-01

    Recent developments in genomic analysis have unveiled the key signaling pathways in human cancer. However, only a limited number of molecular-targeted drugs are applicable for clinical use in gynecologic malignancies. TP53 signaling and phosphatidylinositol 3 kinase pathways are frequently mutated in cancer, and have received much attention as molecular targets in human cancers. In this review, we mainly focus on the functions of these pathways, and discuss the molecular-targeted drugs under clinical trials. The molecular-targeted drugs described in this review include dual phosphatidylinositol 3 kinase/mTOR inhibitors (NVP-BEZ235, DS-7423, SAR245409), an mTOR inhibitor (everolimus), an MEK inhibitor (pimasertib), an autophagy inhibitor (chloroquine), a cyclin-dependent kinases 4/6 inhibitor (PD0332991), and a poly (ADP-ribose) polymerase inhibitor (olaparib). PMID:27094348

  9. Breast cancer in BRCA mutation carriers: medical treatment.

    PubMed

    Milani, Andrea; Geuna, Elena; Zucchini, Giorgia; Aversa, Caterina; Martinello, Rossella; Montemurro, Filippo

    2016-10-01

    About 10% of breast cancers are associated with the inheritance of autosomal dominant breast cancer susceptibility alleles BRCA1 and BRCA2. Until recently, the medical management of BRCA mutation-associated breast cancer has not differed from that of the sporadic breast cancer counterpart. However, there is mounting evidence that this molecular alteration confers sensitivity or resistance to systemic therapies that can be exploited in terms of medical management. For example, studies support the use of platinum salts chemotherapy in BRCA mutated cancers. Moreover, a number of targeted therapies are showing activity in BRCA mutation carriers. Above all, BRCA defective tumor cells are particularly sensitive to Poly(ADP-ribose) polymerase (PARP) inhibitors. This review will summarize the state of the art of the medical treatment of breast cancer in BRCA mutation carriers, with a particular focus on chemotherapies and targeted therapies. PMID:26799758

  10. Repurposing an old drug to improve the safety and use of tissue plasminogen activator for acute ischemic stroke: Minocycline

    PubMed Central

    Hess, David C; Fagan, Susan

    2014-01-01

    There is only 1 US Food and Drug Administration-approved drug for acute ischemic stroke: tissue plasminogen activator (tPA). Due to a short time window and fear of intracerebral hemorrhage (ICH), tPA remains underutilized. There is great interest in developing combination drugs to use with tPA to improve the odds of a favorable recovery and to reduce the risk of ICH. Minocycline is a broad spectrum antibiotic that has been found to be a neuroprotective agent in preclinical ischemic stroke models. Minocycline inhibits matrix metalloproteinase-9, a biomarker for ICH associated with tPA use. Minocycline is also an anti-inflammatory agent and inhibits poly (ADP-ribose) polymerase- 1. Minocycline has been safe and well tolerated in the clinical trials conducted to date. PMID:20575623

  11. Ferulic acid in combination with PARP inhibitor sensitizes breast cancer cells as chemotherapeutic strategy.

    PubMed

    Choi, Young Eun; Park, Eunmi

    2015-03-13

    Homologous-recombination (HR)-dependent repair defective cells are hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Combinations of defective HR pathway and PARP inhibitors have been an effective chemotherapeutic modality. We previously showed that knockdown of the WD40-repeat containing protein, Uaf1, causes an HR repair defect in mouse embryo fibroblast cells and therefore, increases sensitivity to PARP inhibitor, ABT-888. Similarly, here, we show that ferulic acid reduces HR repair, inhibits RAD 51 foci formation, and accumulates γ-H2AX in breast cancer cells. Moreover, ferulic acid, when combined with ABT-888, renders breast cancer cells become hypersensitive to ABT-888. Our study indicates that ferulic acid in combination with ABT-888 treatment may serve as an effective combination chemotherapeutic agent as a natural bioactive compound. PMID:25677620

  12. Olaparib in the management of ovarian cancer

    PubMed Central

    Bixel, Kristin; Hays, John L

    2015-01-01

    Alterations in the homologous repair pathway are thought to occur in 30%–50% of epithelial ovarian cancers. Cells deficient in homologous recombination rely on alternative pathways for DNA repair in order to survive, thereby providing a potential target for therapy. Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, capitalizes on this concept and is the first drug in its class approved for patients with ovarian cancer. This review article will provide an overview of the BRCA genes and homologous recombination, the role of PARP in DNA repair and the biological rationale for the use of PARP inhibitors as cancer therapy, and ultimately will focus on the use of olaparib in the management of ovarian cancer. PMID:26309417

  13. Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

    PubMed Central

    Xia, Gang; Chen, Liangjing; Sera, Takashi; Fa, Ming; Schultz, Peter G.; Romesberg, Floyd E.

    2002-01-01

    The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. PMID:12011423

  14. Efficient production of L-ribose with a recombinant Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new synthetic platform with potential for the production of several rare sugars, with L-ribose being the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression...

  15. Molecular and Cellular Mechanisms of Cardiovascular Disorders in Diabetes.

    PubMed

    Shah, Manasi S; Brownlee, Michael

    2016-05-27

    The clinical correlations linking diabetes mellitus with accelerated atherosclerosis, cardiomyopathy, and increased post-myocardial infarction fatality rates are increasingly understood in mechanistic terms. The multiple mechanisms discussed in this review seem to share a common element: prolonged increases in reactive oxygen species (ROS) production in diabetic cardiovascular cells. Intracellular hyperglycemia causes excessive ROS production. This activates nuclear poly(ADP-ribose) polymerase, which inhibits GAPDH, shunting early glycolytic intermediates into pathogenic signaling pathways. ROS and poly(ADP-ribose) polymerase also reduce sirtuin, PGC-1α, and AMP-activated protein kinase activity. These changes cause decreased mitochondrial biogenesis, increased ROS production, and disturbed circadian clock synchronization of glucose and lipid metabolism. Excessive ROS production also facilitates nuclear transport of proatherogenic transcription factors, increases transcription of the neutrophil enzyme initiating NETosis, peptidylarginine deiminase 4, and activates the NOD-like receptor family, pyrin domain-containing 3 inflammasome. Insulin resistance causes excessive cardiomyocyte ROS production by increasing fatty acid flux and oxidation. This stimulates overexpression of the nuclear receptor PPARα and nuclear translocation of forkhead box O 1, which cause cardiomyopathy. ROS also shift the balance between mitochondrial fusion and fission in favor of increased fission, reducing the metabolic capacity and efficiency of the mitochondrial electron transport chain and ATP synthesis. Mitochondrial oxidative stress also plays a central role in angiotensin II-induced gap junction remodeling and arrhythmogenesis. ROS contribute to sudden death in diabetics after myocardial infarction by increasing post-translational protein modifications, which cause increased ryanodine receptor phosphorylation and downregulation of sarco-endoplasmic reticulum Ca

  16. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

    SciTech Connect

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

    2013-06-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

  17. Cytotoxicity Effects and Apoptosis Induction by Bisbenzylisoquinoline Alkaloids from Triclisia subcordata.

    PubMed

    Uche, Fidelia I; Drijfhout, Falko P; McCullagh, James; Richardson, Alan; Li, Wen-Wu

    2016-09-01

    Triclisia subcordata Oliv (Menispermeaceae) is a medicinal plant traditionally used for the treatment of various diseases in West Africa. The ethanol extract of T. subcordata and its fractions were screened for in vitro anti-ovarian cancer activities using the Sulforhodamine B assay. The crude alkaloids showed the strongest activity in cell growth assays on Ovcar-8 and A2780 cell lines (IC50  < 2.4 µg/mL). A bisbenzylisoquinoline alkaloid-cycleanine was isolated using HPLC and identified by mass spectrometry and nuclear magnetic resonance analyses. The IC50 values of cycleanine and tetrandrine (an alkaloid previously reported from this plant) ranged from 7 to 14 μM on Ovcar-8, A2780, Ovcar-4, and Igrov-1 ovarian cancer cell lines. The IC50 of cycleanine on human normal ovarian surface epithelial cells was 35 ± 1 μM, hinting at modest selectivity toward cancer cells. Both cycleanine and tetrandrine caused apoptosis as shown by activation of caspases 3/7 and cleavage of poly(ADP-ribose) polymerase to form poly(ADP-ribose) polymerase-1 by using western blot analysis. Flow cytometry analyses showed that the percentages of apoptotic cells and cells in subG1 phase increased after exposure of cycleanine and tetrandrine to Ovcar-8 cells for 48 h compared with control. Cycleanine, like its isomer tetrandrine isolated from T. subcordata, could be a potential new anti-ovarian cancer agent acting through the apoptosis pathway. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27270992

  18. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc.

    PubMed

    Cooper, Karen L; King, Brenee S; Sandoval, Monica M; Liu, Ke Jian; Hudson, Laurie G

    2013-06-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. PMID:23523584

  19. Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

    PubMed

    Izumori, K; Rees, A W; Elbein, A D

    1975-10-25

    D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative. PMID:240851

  20. Genome walking by Klenow polymerase.

    PubMed

    Volpicella, Mariateresa; Leoni, Claudia; Fanizza, Immacolata; Rius, Sebastian; Gallerani, Raffaele; Ceci, Luigi R

    2012-11-15

    Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species. PMID:22922302

  1. The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

    NASA Technical Reports Server (NTRS)

    Dworkin, Jason P.; Miller, Stanley L.

    1995-01-01

    If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could

  2. Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA

    PubMed Central

    Koh, Kyung Duk; Balachander, Sathya; Hesselberth, Jay R.; Storici, Francesca

    2015-01-01

    Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed Ribose-seq, a method to capture unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments from yeast Saccharomyces cerevisiae DNA revealed widespread ribonucleotide distribution with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly-synthesized leading strand of nuclear DNA and were present upstream of G+C-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA. PMID:25622106

  3. FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

    PubMed

    Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

    2012-01-01

    Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation. PMID:22470473

  4. FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

    SciTech Connect

    Marshall, Kathryn E.; Robinson, E. W.; Hengel, Shawna M.; Pasa-Tolic, Ljiljana; Roesijadi, Guritno

    2012-03-21

    Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification that enables localization of recombinant proteins in the biosilica cell wall. Our objective was to functionalize diatom biosilica with a reagent-less biosensor with FRET-based imaging capabilities for signaling. The design of the fusion protein conferring these properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the recombinant chimeric protein was first confirmed in E. coli-expressed proteins, prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of CRY showed 95% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica targeting exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 {mu}M and 142.8 {mu}M, respectively. The addition of the silaffin tag for biosilica localization did not influence the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated biosilica in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand binding and conformational change for FRET-signal generation.

  5. Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme.

    PubMed

    Reuter, R; Naumann, M; Bär, J; Miosga, T; Kopperschläger, G

    1998-01-01

    Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure. The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l. For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa. Thus, the native enzyme is composed of four subunits of identical size. The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S. cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene. On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented. PMID:9763699

  6. Fragmentation of deprotonated d-ribose and d-fructose in MALDI--Comparison with dissociative electron attachment

    NASA Astrophysics Data System (ADS)

    Bald, Ilko; Flosadóttir, Helga D.; Kopyra, Janina; Illenberger, Eugen; Ingólfsson, Oddur

    2009-02-01

    We present a detailed, collaborative study on the fragmentation of deprotonated native d-ribose and d-fructose and the isotopically labelled 1-13C-d-ribose, 5-13C-d-ribose and C-1-d-d-ribose. The fragmentation is studied in a matrix assisted laser desorption/ionization time of flight mass spectrometer (MALDI ToF MS), both in in-source decay (ISD) and post-source decay (PSD) mode and compared with fragmentation through dissociative electron attachment (DEA). Fragmentation of deprotonated monosaccharides formed in the MALDI process, as well as their transient molecular anions formed upon electron attachment are characterized by loss of different numbers of H2O and CH2O units. Two different fragmentation pathways leading to cross-ring cleavage are identified. Metastable decay of deprotonated d-ribose proceeds either via an X-type cleavage yielding fragment anions at m/z = 119, 100 and 89, or via an A-type cleavage resulting in m/z = 89, 77 and 71. A fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in in-source decay is dominated by X-type cleavage leading mainly to m/z = 100 and 71. For dissociative electron attachment to d-ribose a sequential dissociation was identified that includes metastable decay of the dehydrogenated molecular anion leading to m/z = 89. All other fragmentation reactions in DEA to d-ribose are likely to proceed directly and on a faster timescale (below 400 ns).

  7. Cellular protection during oxidative stress: a potential role for D-ribose and antioxidants.

    PubMed

    Addis, Paul; Shecterle, Linda M; St Cyr, John A

    2012-09-01

    A healthy cellular system involves the maintenance of an intracellular metabolic balance. Reactive oxygen species (ROS) are constantly produced as a normal product of cellular metabolism; however, during situations of cellular stress, these levels can increase dramatically with the potential to cause deleterious cellular structural and/or functional consequences. There is a significant elevation in these ROS following stressful situations, such as ischemia, hypoxia, high-intensity exercise, and in many diseases. To combat these ROS, neutralizing endogenous enzymes, as well as exogenous antioxidants, can aid in minimizing their potential untoward cellular effects. Exogenous reducing antioxidant agents, such as vitamin C and/or E, play a role in addressing these formed species; however, recent research has suggested that fruit seed extracts may provide additional cellular benefits beyond their antioxidant features. Furthermore, supplemental D-ribose enhances the recovery of high-energy phosphates following stress and appears to potentially offer additional benefits by reducing radical formation. Specifically, during periods of hypoxia/ischemia, supplemental D-ribose may play an inhibitory role in the breakdown of adenine nucleotides, influencing the subsequent formation of xanthine and uric acid compounds; and thereby affecting the release of superoxide anion radicals. The combination of D-ribose with reducing antioxidants may provide a more optimal state of cellular protection during and following times of oxidative stress. PMID:22891990

  8. 2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family

    PubMed Central

    Werner, Maria; Purta, Elżbieta; Kaminska, Katarzyna H.; Cymerman, Iwona A.; Campbell, David A.; Mittra, Bidyottam; Zamudio, Jesse R.; Sturm, Nancy R.; Jaworski, Jacek; Bujnicki, Janusz M.

    2011-01-01

    The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained. PMID:21310715

  9. A bridge to transcription by RNA polymerase.

    PubMed

    Kaplan, Craig D; Kornberg, Roger D

    2008-01-01

    A comprehensive survey of single amino-acid substitution mutations critical for RNA polymerase function published in Journal of Biolo