Presence Of Poly(ADP-ribose) Polymerase-1 Research Articles

ZSCAN4 interacts with PARP1 to promote DNA repair in mouse embryonic stem cells

BackgroundIn eukaryotic cells, DNA double strand breaks (DSB) are primarily repaired by canonical non-homologous end joining (c-NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ). Zinc finger and SCAN domain containing 4 (ZSCAN4), sporadically expressed in 1–5% mouse embryonic stem cells (mESCs), is known to regulate genome stability by promoting HR.ResultsHere we show that ZSCAN4 promotes DNA repair by acting with Poly (ADP-ribose) polymerase 1 (PARP1), which is a key member of the alt-NHEJ pathway. In the presence of PARP1, ZSCAN4-expressing mESCs are associated with lower extent of endogenous or chemical induced DSB comparing to ZSCAN4-negative ones. Reduced DSBs associated with ZSCAN4 are abolished by PARP1 inhibition, achieved either through small molecule inhibitor or gene knockout in mESCs. Furthermore, PARP1 binds directly to ZSCAN4, and the second ⍺-helix and the fourth zinc finger motif of ZSCAN4 are critical for this binding.ConclusionsThese data reveal that PARP1 and ZSCAN4 have a protein–protein interaction, and shed light on the molecular mechanisms by which ZSCAN4 reduces DSB in mESCs.

Renewable electrochemical sensor for PARP-1 activity detection based on host-guest recognition

Poly (ADP-ribose) polymerase-1 (PARP-1) was defined as a new biomarker, which has achieved wide attention in recent years. In this work, we designed a renewable electrochemical sensor based on host-guest recognition for the detection of PARP-1 activity. Mono-(6-Mercapto-6-deoxy)-beta-Cyclodextrin (SH-β-CD) was modified on the electrode surface to recognize the trans-azobenzene labeled dsDNA (Abz-dsDNA). In the presence of PARP-1, PAR with abundant PO43- was generated and reacted with MoO42- to form PMo12O403-, producing strong current. The proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azobenzene from trans to cis structure, allowing the electrode to be recycled. The sensor realized the linear detection of PARP-1, ranging from 0.01 U to 1.0 U with a detection limit of 0.008 U, which is comparable to results from reported methods. It is expected to be a potential tool for clinical detection because of its high sensitivity and selectivity.

Altered Cellular Responses As Biomarkers for Fluoranthene Exposure to the Bone Marrow-Mesenchymal Stem Cells: Implications for Stem Cell-Based in Vitro Models That Help Determine the Associated Hematotoxicity

Background: Fluoranthene is a member of the polycyclic aromatic hydrocarbon family, comprising ubiquitous environmental pollutants and potent mutagens. Fluoranthene has been classified by the International Agency for Research on Cancer as a group 3 carcinogens, whose carcinogenicity has not been fully determined in humans. Moreover, the stem cell toxicity and global hematotoxicity associated with fluoranthene exposure have not been thoroughly studied in the bone marrow-mesenchymal stem cells (BM-MSCs). In this study, we determined whether fluoranthene-induced cellular responses could be used as biomarkers for the examination of BM-MSC dysfunction, and suggested the possible use of BM-MSCs for monitoring the acute hematotoxicity caused by environmental hazards.Materials and Methods: Apreviously published protocol was used for the isolation and characterization of BM-MSCs. Fluoranthene was added to the cell culture medium in the range of 25–500 µM. The cellular levels of hydrogen peroxide, indicating the presence of reactive oxygen species, were measured using an enzyme immunoassay. The mitochondrial mass, membrane potential, and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, MitoTracker Red probes, and real time PCR, respectively. A proteomic analysis of the mitochondrial-rich cytoplasmic fraction was performed using nano-LC-ESI-MS/MS, BioWorksBrowser, and the SEQUEST search engines. Quantitative mRNA and immunoblot measurements were used to further confirm the altered mRNA expression as well as to determine the levels of cellular proteins obtained from the proteomic analysis.Results: After exposure to fluoranthene, the BM-MSCs showed a marked reduction in cell number, and the viability decreased substantially after two days of exposure. BM-MSCs that were not treated with fluoranthene remained compact and spindle-shaped. These cells remained tightly attached to each other and to the substrate. In general, a direct exposure of fluoranthene depressed the proliferative capacity and altered the cell morphology of BM-MSCs. The cells detached from the subsurface, and cell-to-cell attachments were also lost. The viability significantly decreased after two days of fluoranthene exposure. The mtDNA copy number and the mass showed a rapid elevation after a 5-day exposure to fluoranthene. Hundreds of cellular proteins in the mitochondria-rich cytoplasmic fraction were markedly deregulated in cells treated with fluoranthene. The protein expression levels of poly [ADP-ribose] polymerase 1 (PARP-1), elongation factor 1-gamma, heat shock 70 kDa protein 1A/1B, heterogeneous nuclear ribonucleoproteins A2/B1 isoform B1, ATP-dependent RNA helicase DDX5, and T-complex protein 1 subunit theta were upregulated more than five-fold in cells treated with fluoranthene than in untreated cells. A significant (more than 2-fold) down-regulation in the cellular levels of the proteins myosin-9, protein ALO17 isoform 1, filamin-C isoform b, Na/K-transporting ATPase subunit alpha-1, nuclear pore membrane glycoprotein 210, and DNA-dependent protein kinase catalytic subunit isoform 2 was observed after a similar fluoranthene treatment. The presence of PARP-1 was further confirmed using mRNA analysis.Conclusion: This study investigated the global cellular responses after exposure to fluoranthene: PARP-1 was recognized as a notable biomarker for monitoring the PAH-induced hematotoxicity. In summary, BM-MSCs are promising candidates for the development of unique in vitro model systems for predicting fluoranthene-associated hematotoxicity and general toxicity in humans.Keywords: Fluoranthene, genotoxicity, hematotoxicity, bone marrow-mesenchymal stem cells [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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.

Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD+ and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.

Abstract C297: PARP1 and BIN1 cooperate to act as a novel co-repressor for E2F1.

Abstract Background: Bridging integrator 1 (BIN1) is a transcriptional co-repressor and inhibits oncogenic activities of c-MYC and E1A oncoproteins1. BIN1 inhibits c-MYC through direct interaction, whereas the mechanism by which BIN1 attenuates the oncogenic transformation mediated by adenovirus E1A remains elusive. Since BIN1 does not physically interact with E1A, we hypothesized that BIN1 diminishes a cellular effector, which can be activated by E1A and is essential for E1A transformation. One of the well-recognized cellular effectors for E1A transformation is the E2F1 transcription factor. Although retinoblastoma (RB) protein is an authentic E2F1 inhibitor, loss of RB does not immediately result in cancer development, suggesting that an anti-E2F1 function, which is independent of RB, compensates for RB deficiency. BIN1 was recently shown to interact with poly (ADP-ribose) polymerase1 (PARP1), a component of transcriptional complex2. Given that PARP1 also interacts with E2F1, we hypothesized functional cross talk between BIN1 and PARP1 to curb E2F1 activity. Experimental Procedures: E2F1 activity was determined using an adenovirus-driven E2F1-sensitive luciferase reporter vector, E2A-Luc. Protein-protein interaction was studied by co-immunoprecipitation followed by Western blot analysis. Chromatin immunoprecipitation assay was used to demonstrate protein-DNA interaction. Colony formation and foci formation assays were performed to investigate BIN1-mediated tumor suppression in the presence and absence of PARP1. Results: We discovered that BIN1 physically interacts with E2F1 and inhibits its transactivation. Interestingly, depletion of PARP1 released endogenous E2F1 activity, regardless of the status of RB expression. Furthermore, BIN1 failed to inhibit E2F1 activity in the absence of PARP1, but RB does not need PARP1 to inhibit E2F1 activity. Chromatin immunoprecipitation assays suggested that BIN1 and PARP1 co-existed on an E2F-responsive promoter. Moreover, in the absence of PARP1, the binding affinities of both BIN1 and E2F1 to the promoter were significantly reduced. In addition, only in the presence of PARP1, ectopically expressed BIN1 inhibited tumor colony formation and HPV16 E7 oncoprotein-dependent cellular transformation. Our results suggest that BIN1 and PARP1 cooperate to inhibit E2F1 activity and suppress oncogenic transformation. Conclusion: We conclude that BIN1 is a novel E2F1 corepressor in cooperation with PARP1. BIN1-PARP1 interaction may serve as a safety device to control deregulated E2F1 activity due to RB loss. This study was supported by NIH R01CA140379 (to D.S.). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C297. Citation Format: Alpana Kumari, Slovénie Pyndiah, Erica K. Cassimere, Daitoku Sakamuro. PARP1 and BIN1 cooperate to act as a novel co-repressor for E2F1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C297.

Abstract 2968: The functional relationship between CHFR and PARP-1 controls the antephase checkpoint and tumor development

Abstract The mitotic checkpoint gene CHFR is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase of Aurora A and HDAC1, resulting in the degradation of those proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Poly (ADP-ribose) polymerase-1 (PARP-1), which catalyzes polyADP-ribosylation, was newly identified as a CHFR interacting protein. CHFR promoted polyubiquitination of PARP-1. Conversely, PARP-1 poly(ADP-ribosyl)ated CHFR. In addition, we mapped the region of both CHFR and PARP-1 required for the interaction. Because CHFR delays entry into metaphase in response to mitotic stress, we investigated whether PARP-1 is involved in the mitotic checkpoint. In the presence of PARP-1, CHFR arrested the cell cycle. On the other hand, in the absence of PARP-1, CHFR had no effect on the cell cycle arrest. Additionally, we found the knockdown of PARP-1 resulted in cell cycle arrest itself. As overexpression of PARP-1 has been reported in several human tumors, our findings suggest the functional relationships between CHFR and PARP-1 could play an important role in regulation of cell cycle and tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2011-2968

Abstract 688: Acetylation of poly-ADP-ribose polymerase and Ku70 by histone deacetylase inhibitors promote abnormal binding to DNA double strand breaks and decrease repair efficiency in leukemia cells

Abstract Histone Deacetylase inhibitors (HDACi) affect gene expression through acetylation of histone and non-histone proteins which could alter their cellular activity. We have previously shown that HDACi trigger DNA damage responses in leukemia cells, and actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist longer compared with normal cells. The mechanism by which this DNA damage occurs is still unknown. Non Homologous End Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. We have recently shown that NHEJ activity is abnormal in myeloid leukemias in that classical NHEJ that is Ku and DNA-PKcs dependent, is down regulated, and compensatory repair of DSBs occurs via alternative (Alt) NHEJ pathways involving Poly-ADP-Ribose Polymerase-1 (PARP-1) and DNA LigaseIIIα. Alt NHEJ is known to generate large deletions and translocations in cancers and leukemias. To determine whether HDACi may decrease the capacity of leukemic cells to repair DSBs by direct acetylation of NHEJ proteins, K562 and HL60 leukemia cell lines were treated with Trichostatin A (TSA, 300nM for 1 and 6 hours) followed by immunoprecipitation and western blot analysis for acetylated NHEJ proteins. TSA treatment of K562 and HL60 show differential acetylation of classical NHEJ protein Ku70 and Alt NHEJ protein PARP-1, compared with controls. To investigate whether TSA treatment alters the binding of Ku70 and PARP-1 to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with DRNeo that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after TSA treatment while Ku70 is not. To determine whether DSB repair activity is altered after HDACi treatment, we performed a plasmid based NHEJ repair assay in our leukemia cell lines after treatment with TSA. Both leukemia cell lines demonstrate a significant decrease in the capacity to repair DSBs following HDACi treatment. The same result can be obtained by the use of another HDACi entinostat. These data suggest that HDACi treatment leads to an increased presence of PARP-1 at DSBs, preventing subsequent critical repair steps in leukemia cells. Importantly, AML cells from patients treated with entinostat in vivo show significantly increased co-localization of PARP-1 and γH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Overall, our results suggest that HDACi result in both a physical and functional alteration of proteins participating in both classical and Alt NHEJ, leading to a loss of binding of Ku70 and an abnormal persistence of PARP-1 at DSBs. Our data supports the idea that acetylated PARP-1 prevents repair of DSBs by Alt NHEJ, leading to a persistence of DSBs in leukemia cells treated with HDACi. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 688.

Poly(ADP‐ribose) polymerase‐1 modulation of in vivo response of brain hypoxia‐inducible factor‐1 to hypoxia/reoxygenation is mediated by nitric oxide and factor inhibiting HIF

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that once activated by genotoxic agents, modulates its own activity and that of several other nuclear proteins. The absence or pharmacological inhibition of this protein has been proven to be beneficial in the treatment of different diseases involving a hypoxic situation. We previously reported that PARP-1 modulates the hypoxia-inducible factor-1 (HIF-1) response in vitro, but this effect has not yet been demonstrated in vivo. The brain is especially susceptible to hypoxic injury, and the present study demonstrates that PARP-1 plays a major role in the post-hypoxic response of HIF-1alpha in the cerebral cortex. Immediate post-hypoxic HIF-1alpha accumulation was higher in the presence of PARP-1, and this differential response was mediated by nitric oxide and to a lesser extent, reactive oxygen species. PARP-1 was also found to induce a more rapid but less sustained HIF-1 transcriptional activity by up-regulating the factor inhibiting HIF. The implication of PARP-1 in these results was further demonstrated by pharmacologically inhibiting PARP in wild-type mice. In conclusion, our data suggest that PARP-1 has an important regulatory role in the in vivo response of brain HIF-1 to hypoxia/reoxygenation.

Poly(ADP-ribose) polymerase-1 inhibits strand-displacement synthesis of DNA catalyzed by DNA polymerase beta.

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.

Intra-mitochondrial Poly(ADP-ribosylation) Contributes to NAD+ Depletion and Cell Death Induced by Oxidative Stress

Poly(ADP-ribosylation), primarily via poly(ADP-ribose) polymerase-1 (PARP-1), is a pluripotent cellular process important for maintenance of genomic integrity and RNA transcription in cells. However, during conditions of oxidative stress and energy depletion, poly(ADP-ribosylation) paradoxically contributes to mitochondrial failure and cell death. Although it has been presumed that poly(ADP-ribosylation) within the nucleus mediates this pathologic process, PARP-1 and other poly(ADP-ribosyltransferases) are also localized within mitochondria. To this end, the presence of PARP-1 and poly(ADP-ribosylation) were verified within mitochondrial fractions from primary cortical neurons and fibroblasts. Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential (DeltaPsi(m)), NAD(+) content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress. Treatment with liposomal NAD(+) also preserved DeltaPsi(m) and cellular respiration during oxidative stress. Furthermore, inhibition of poly(ADP-ribosylation) prevented intranuclear localization of apoptosis-inducing factor and protected neurons from excitotoxic injury; and PARP-1 null fibroblasts were protected from oxidative stress-induced cell death. Collectively these data suggest that poly(ADP-ribosylation) compartmentalized to the mitochondria can be converted from a homeostatic process to a mechanism of cell death when oxidative stress is accompanied by energy depletion. These data implicate intra-mitochondrial poly(ADP-ribosylation) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.

Solubilization of enzyme forming ADPR polymer from NAD

Poly (ADP-ribose) polymerase-1 (PARP-1) was defined as a new biomarker, which has achieved wide attention in recent years. In this work, we designed a renewable electrochemical sensor based on host-guest recognition for the detection of PARP-1 activity. Mono-(6-Mercapto-6-deoxy)-beta-Cyclodextrin (SH-β-CD) was modified on the electrode surface to recognize the trans-azobenzene labeled dsDNA (Abz-dsDNA). In the presence of PARP-1, PAR with abundant PO43- was generated and reacted with MoO42- to form PMo12O403-, producing strong current. The proposed method avoided the unspecific adsorption effectively and improved the detection accuracy. Under UV irradiation, Abz-dsDNA was removed from the electrode surface because of the configuration change of azobenzene from trans to cis structure, allowing the electrode to be recycled. The sensor realized the linear detection of PARP-1, ranging from 0.01 U to 1.0 U with a detection limit of 0.008 U, which is comparable to results from reported methods. It is expected to be a potential tool for clinical detection because of its high sensitivity and selectivity.