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

Background-Quenched Aggregation-Induced Emission through Electrostatic Interactions for the Detection of Poly(ADP-ribose) Polymerase-1 Activity.

Poly(ADP-ribose) polymerase-1 (PARP1) is a potential biomarker and therapeutic target for cancers that can catalyze the poly-ADP-ribosylation of nicotinamide adenine dinucleotide (NAD+) onto the acceptor proteins to form long poly(ADP-ribose) (PAR) polymers. Through integration with aggregation-induced emission (AIE), a background-quenched strategy for the detection of PARP1 activity was designed. In the absence of PARP1, the background signal caused by the electrostatic interactions between quencher-labeled PARP1-specitic DNA and tetraphenylethene-substituted pyridinium salt (TPE-Py, a positively charged AIE fluorogen) was low due to the fluorescence resonance energy transfer effect. After poly-ADP-ribosylation, the TPE-Py fluorogens were recruited by the negatively charged PAR polymers to form larger aggregates through electrostatic interactions, thus enhancing the emission. The detection limit of this method for PARP1 detection was found to be 0.006 U with a linear range of 0.01~2 U. The strategy was used to evaluate the inhibition efficiency of inhibitors and the activity of PARP1 in breast cancer cells with satisfactory results, thus showing great potential for clinical diagnostic and therapeutic monitoring.

Poly(ADP-ribose) polymerase 1 in genome-wide expression control in Drosophila

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme involved in DNA repair and transcription regulation, among other processes. Malignant transformations, tumor progression, the onset of some neuropathies and other disorders have been linked to misregulation of PARP-1 activity. Despite intensive studies during the last few decades, the role of PARP-1 in transcription regulation is still not well understood. In this study, a transcriptomic analysis in Drosophila melanogaster third instar larvae was carried out. A total of 602 genes were identified, showing large-scale changes in their expression levels in the absence of PARP-1 in vivo. Among these genes, several functional gene groups were present, including transcription factors and cytochrome family members. The transcription levels of genes from the same functional group were affected by the absence of PARP-1 in a similar manner. In the absence of PARP-1, all misregulated genes coding for transcription factors were downregulated, whereas all genes coding for members of the cytochrome P450 family were upregulated. The cytochrome P450 proteins contain heme as a cofactor and are involved in oxidoreduction. Significant changes were also observed in the expression of several mobile elements in the absence of PARP-1, suggesting that PARP-1 may be involved in regulating the expression of mobile elements.

Poly(ADP-ribose) polymerase-1 depletion enhances the severity of inflammation in an imiquimod-induced model of psoriasis.

Poly(ADP-ribose) polymerase-1 (PARP1) is a pro-inflammatory protein, whose pro-inflammatory properties were demonstrated in human. The pro-inflammatory properties of PARP1 were shown in Th1- and Th2-mediated inflammatory pathologies, but not Th17-mediated inflammation. Thus, we studied the role of PARP1 in the imiquimod-induced model of psoriasis. To our surprise, in imiquimod-induced psoriasis, PARP1 acted as an anti-inflammatory factor and its genetic deletion exacerbated symptoms. We showed that in the absence of PARP1, the epidermis thickened and the number of TUNEL-positive cells decreased in the epidermis. These data indicate programmed cell death is decreased in keratinocytes. Changes in involucrin expression suggest that keratinocyte differentiation is hampered. Furthermore, epidermal expression of IL6 increased in the psoriasiform lesions of PARP1 knockout mice, suggesting that the inflammatory response is also derailed in the absence of PARP1. Finally, we showed that PARP1 expression is reduced in human psoriatic lesions compared with control skin samples. In imiquimod-treated HPV-KER keratinocytes, PARP inhibition recapitulated the in vivo findings, namely keratinocyte hyperproliferation; furthermore, the mRNA expression of psoriasis-associated cytokines (IL6, IL1β, IL8, IL17 and IL23A) was also induced. The inhibition of TRPV1 abrogated the effects of the combined imiquimod+PARP inhibitor treatment.

Evidence for Decreased Nucleolar PARP-1 as an Early Marker of Cognitive Impairment.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that regulates gene expression through poly(ADP)-ribosylation, resulting in the loosening of chromatin structure. PARP-1 enzymatic activity has been shown to be necessary for the expression of several genes required for memory formation and consolidation. Previously, we showed that nucleolar PARP-1 is significantly decreased in hippocampal pyramidal cells in Alzheimer's disease (AD). We proposed that the displacement of PARP-1 from the nucleolus results in downregulation of new rRNA expression and ribosome biogenesis, leading to cognitive impairment. To further investigate the relationship between nucleolar PARP-1 and memory impairment, we examined PARP-1 expression in the hippocampi of individuals with mild cognitive impairment (MCI) compared to control and AD cases. We used immunohistochemical techniques to examine the nucleolar distribution of PARP-1 in the Cornu Ammonis (CA region) of the hippocampus. PARP-1 positive cells were then scored for the presence or absence of PARP-1 in the nucleolus. We found a significant decrease of PARP-1 staining in the nucleolar compartment of hippocampal pyramidal cells in MCI compared with Control and AD. When the four CA (CA1-4) regions were considered separately, only the CA1 region showed significant differences in nucleolar PARP-1 with Control > AD > MCI cases. Categorization of nucleolar PARP-1 into “distinct” and “diffuse” groups suggest that most of the changes occur within the distinct group. In addition, measurements of the nucleolar diameter of nucleolar PARP-1 positive cells in CA2 and CA4 showed Control > MCI. Thus, MCI cases had a lower percentage of PARP-1 nucleolar positive cells in CA1 and smaller nucleolar diameters in CA2 and CA4, compared to Control. Our data suggest that disruption of nucleolar form and function is an early and important step in the progression of cognitive impairment.

Enrichment of Cxcl12 promoter with TET2: A possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1

Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation (TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3) and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173020]

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

Poly(ADP-ribose) polymerase 1 and its inhibitors in tumor treatment

Poly( ADP-ribose) polymerase 1 (PARP-1) plays an essential role in DNA repair and maintenance of homeostasis and genome stability. Increased PARP-1 activity has been reported in various forms of cancer. Thus the absence of PARP-1 or the use of its inhibitors depresses DNA repair function, sensitizes cancer cells to DNA damage and enhances the therapeutic effect of radio-or chemotherapy. PARP-1 is potentially a therapeutic target of malignant tumors. Key words: Poly(ADP-ribose) polymerases; DNA repair enzymes; Neoplasms; Poly (ADP-ribose) polymerases inhibitors

Poly(ADP-Ribose) Polymerase-1 Regulates the Progression of Autoimmune Nephritis in Males by Inducing Necrotic Cell Death and Modulating Inflammation

Necrotic lesions and necrotic cell death characterize severe autoimmune nephritides, and contribute to local inflammation and to progression of the disease. Poly(ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme, is involved in the induction of necrosis and is a key player in the acute and chronic inflammation. Therefore, we hypothesized that PARP-1 controls the severity of nephritis by mediating the induction of necrosis in the kidney. We used lupus and anti-glomerular basement membrane models of nephritis to determine the effects of PARP-1 on the inflammatory response in the kidney. We show in this study that PARP-1 is indeed activated during the course of glomerulonephritis. We also show that the absence of PARP-1 or its pharmacological inhibition results in milder nephritis, with lower blood urea nitrogen levels, reduced necrotic lesions, and higher survival rates. The relevance of PARP-1 showed a strong male sex specificity, and treatment of male mice with 17beta-estradiol prolonged their survival during the course of nephritis. PARP-1 also regulated TNF-alpha expression and up-regulation of adhesion molecules, further supporting a role of PARP-1 in the inflammatory process within the kidney. Our results demonstrate that PARP-1 activation and consequent necrotic cell death play an important role in the pathogenesis of male nephritis, and suggest that PARP-1 can be a novel therapeutic target in glomerulonephritis.

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.

Poly(ADP-ribose)polymerase Activation Mediates Lung Epithelial Cell Death In Vitro but Is Not Essential in Hyperoxia-Induced Lung Injury

Hyperoxia induces extensive DNA damage and lung cell death by apoptotic and nonapoptotic pathways. We analyzed the regulation of Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme activated by DNA damage, and its relation to cell death during hyperoxia in vitro and in vivo. In lung epithelial-derived A549 cells, which are known to die by necrosis when exposed to oxygen, a minimal amount of PARP-1 was cleaved, correlating with the absence of active caspase-3. Conversely, in primary lung fibroblasts, which die mainly by apoptosis, the complete cleavage of PARP-1 was concomitant to the induction of active caspase-3, as assessed by Western blot and caspase activity. Blockade of caspase activity by Z-VAD reduced the amount of cleaved PARP-1 in fibroblasts. Hyperoxia induced PARP activity in both cell types, as revealed by poly-ADP-ribose accumulation. In A549 cells, the final outcome of necrosis was dependent on PARP activity because it was prevented by the PARP inhibitor 3-aminobenzamide. In contrast, apoptosis of lung fibroblasts was not sensitive to 3-aminobenzamide and was not affected by PARP-1 deletion. In vivo, despite evidence of PARP activation in hyperoxia-exposed mouse lungs, absence of PARP-1 did not change the extent of lung damage, arguing for redundant oxidative stress-induced cell death pathways.

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.

Crosstalk between PARP-1 and NF-kappaB modulates the promotion of skin neoplasia.

Poly (ADP-ribose) polymerase-1 (PARP-1)-deficient mice are protected against septic shock, type I diabetes, stroke and inflammation. It is now accepted that inflammation and related events, such as activation of NF-kappaB, are key components in the initiation and progression of epithelial cancer and in particular in the neoplastic transformation of keratinocytes and skin carcinogenesis. Here, we report that PARP-1-deficient mice display a strikingly reduced susceptibility to skin carcinogenesis. In parp-1(-/-) mice, development of papilloma-like premalignant lesions induced with DMBA and TPA, is strongly delayed and the final number of tumor-bearing mice and total tumor number were significantly reduced. In addition, epidermis of parp-1(-/-) mice did not show increased proliferation rates after treatment with carcinogen. Deregulated NF-kappaB is a hallmark for tumorigenesis together with the concomitant release of early inflammatory mediators. In the absence of PARP-1, NF-kappaB activation and induction kappaB-target genes did not take place during the promotion of tumor development. These results suggest that PARP-1 abolition impairs the promotion of skin carcinogenesis interfering with the activation of NF-kappaB and might have an important implication in targeting PARP-1 as a new antineoplastic therapeutic approach.

Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated with various pathophysiological conditions. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction could be related to PARP-1 overactivation, NAD(+) consumption and ATP depletion. An alternative way could involve transcription regulation. By using high-density microarrays, we examined early tumor necrosis factor alpha (TNF-alpha)-stimulated gene expression profiles in PARP-1(+/+) and PARP-1(-/-) murine heart endothelial cells. TNF-alpha modulated a significant number of genes in both cell types. We have identified a set of genes whose expression in response to TNF-alpha is modulated by PARP-1, whereas the expression of others is PARP-1-independent. Up-regulation of several genes involved in the inflammatory response is hampered in the absence of PARP-1. Moreover, NF-kappaB-dependent transcriptional activation is partially inhibited in PARP-1(-/-) compared to PARP-1(+/+) cells. However, we found that PARP-1 might also silence transcription of several NF-kappaB target genes. Overall, our results show that PARP-1 is regulating the expression of genes by the endothelial cells both in a positive and a negative fashion, with the final effects depending on the gene. Individual studies of these genes are now necessary to clarify the intrinsic mechanism by which PARP-1 is controlling transcription and thereby finding out different therapeutic approaches involving PARP-1.