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PubMed2000

Effects of transient global ischemia and kainate on poly(ADP-ribose) polymerase (PARP) gene expression and proteolytic cleavage in gerbil and rat brains.

Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg/kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.

Animals↗
PubMed2005

Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1-/- than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

Alkylating Agents↗
PubMed2006

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 nonhomologous 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.

1-Naphthylamine↗
PubMed2007

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Amino Acid Sequence↗
PubMed2000

Characterization of sPARP-1. An alternative product of PARP-1 gene with poly(ADP-ribose) polymerase activity independent of DNA strand breaks.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that catalyzes the synthesis of poly(ADP-ribose) (pADPr) from its substrate NAD(+) upon binding to DNA strand breaks. Poly(ADP-ribosyl)ation has been implicated in many cellular processes including replication, transcription, and the maintenance of genomic stability. However, studies with mice and cells lacking PARP-1 reveal a critical role for the enzyme in the maintenance of genomic integrity only. Recently, a significant level of poly(ADP-ribose) polymerase activity has been detected in fibroblasts derived from mice lacking PARP-1 following treatment with genotoxic agents (Shieh, W. M., Amé, J-C., Wilson, M. V., Wang, Z-Q., Koh, D. W., Jacobson, M. K., and Jacobson, E. L. (1998) J. Biol. Chem. 273, 30069-30072). We have isolated a cDNA that originates from PARP-1 (-/-) fibroblasts and encodes a polypeptide of 493 amino acid residues bearing poly(ADP-ribose) polymerase activity. This protein, that we named sPARP-1 for short poly(ADP-ribose) polymerase-1, has a calculated mass of 55.3 kDa and is identical in deduced amino acid sequence to the catalytic domain of PARP-1. Radiation hybrid analysis assigned the sPARP-1 gene to the chromosome 1H5-H6 in an immediate proximity to the known location of PARP-1 gene, indicating that sPARP-1 and PARP-1 are most probably products of the same gene. Active sPARP-1 is present in both PARP-1 (+/+) and PARP-1 (-/-) cells as demonstrated by activity-Western blotting and immunostaining using a specific antibody developed against sPARP-1. Like PARP-1, sPARP-1 is localized in the cell nucleus, uses NAD(+) as a substrate and is inhibited by nicotinamide analogues. sPARP-1 produces pADPr of similar length and structure to that of PARP-1. However, contrary to PARP-1, sPARP-1 does not require DNA strand breaks for its activation, although it is stimulated following genotoxic treatments.

Alternative Splicing↗
PubMed1999

Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts.

The interaction between poly(ADP-ribose) polymerase (PARP) and the product of the tumor suppressor gene p53 has been described previously. Here, we have investigated whether PARP deficiency may affect the expression and regulation of wild-type (wt) p53. For this purpose, we have used immortalized cells derived from wt and PARP knockout mice. We have found a clearly reduced basal level of PAb421 immunoreactive wt p53 protein in PARP-deficient cells. The monoclonal antibody PAb421 is known to recognize an epitope in the COOH terminus of normally spliced p53 protein. Under indirect immunofluorescence, this antibody stained nuclei in normal but not in PARP-deficient cells. Despite marked reduction of wt p53 protein in PARP knockout cells, no significant difference of the p53 transcription rate was observed between wt and PARP-deficient cells. Interestingly, in both cell types, an additional p53 transcript representing the alternatively spliced (AS) p53 form was detected. Because of its reactivity with different specific anti-p53 antibodies, we have determined that the p53 protein present in PARP knockout mouse cells possesses characteristic features of AS p53. Our results clearly show that PARP-deficient cells constitutively express the AS form of wt p53 and indicate that the regularly spliced p53 is extremely unstable in the absence of PARP. Moreover, PARP-/- cells fail to transactivate p53-responsive genes. Treatment of PARP-/- cells with genotoxic agents primarily leads to the activation of AS p53 protein.

Animals↗
PubMedDate not supplied

Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation.

Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.

Apoptosis↗
PubMed1999

Trypanosoma brucei: cis-acting sequences involved in the developmental regulation of PARP expression.

The procyclic acidic repetitive protein (PARP or procyclin) of the parasitic protozoan Trypanosoma brucei is a developmentally regulated protein that shows extreme differences in its level of expression in different stages of the parasite's life cycle. Specifically, it is the major surface protein in the procyclic (insect) stage and, although the PARP gene is being actively transcribed in the mammalian bloodstream stage, there is no detectable PARP mRNA or protein in these cells. The 3'-untranslated region (UTR) of PARP, as well as other trypanosome genes, has the ability to confer the appropriate developmental regulation pattern onto chimeric reporter genes. To understand the mechanism of posttranscriptional regulation, selective replacement mutagenesis of the PARP mRNA 3'UTR was done to identify the cis-acting sequences involved in the down-regulation of this mRNA in bloodstream-form T. brucei. Transient transformation of constructs containing the PARP promoter and 5'UTR, the beta-glucuronidase coding region, and the selectively mutagenized or unaltered PARP 3'UTR were performed in procyclic and bloodstream T. brucei. The results of the reporter gene assays on the transformed cells indicate that there are at least two elements in the PARP 3'UTR which in bloodstream cells are involved in regulation of PARP expression and which appear to function as negative elements. In procyclic cells, there are two regions in which mutagenesis indicates positive cis-regulatory sequences, one of which has been previously defined (A. Hehl et al., 1994, Proc. Natl. Acad. Sci. USA 91, 370-374). These results indicate that multiple cis-acting elements within the PARP 3'UTR are involved in the developmental regulation of PARP expression and that regulation is controlled in a complex manner, presumably involving several cellular trans-acting factors.

Animals↗
PubMed2002

Potential clinical applications of poly(ADP-ribose) polymerase (PARP) inhibitors.

Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD(+)to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, that presently includes six members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress (oxygen radicals, ionizing radiations and monofunctional alkylating agents). Due to its involvement either in DNA repair or in cell death, PARP-1 is regarded as a double-edged regulator of cellular functions. In fact, when the DNA damage is moderate, PARP-1 participates in the DNA repair process. Conversely, in the case of massive DNA injury, elevated PARP-1 activation leads to rapid NAD(+)/ATP consumption and cell death by necrosis. Excessive PARP-1 activity has been implicated in the pathogenesis of numerous clinical conditions such as stroke, myocardial infarction, shock, diabetes and neurodegenerative disorders. PARP-1 could therefore be considered as a potential target for the development of pharmacological strategies to enhance the antitumor efficacy of radio- and chemotherapy or to treat a number of clinical conditions characterized by oxidative or NO-induced stress and consequent PARP-1 activation. Moreover, the discovery of novel functions for the multiple members of the PARP family might lead in the future to additional clinical indications for PARP inhibitors.

Animals↗
PubMed2005

Clinical perspectives of PARP inhibitors.

Poly(ADP-ribose) polymerase (PARP) activation plays a role in the pathogenesis of various cardiovascular and inflammatory diseases. At the same time, PARP activation is also relevant for the ability of cells to repair injured DNA. Thus, depending on the circumstances, pharmacological inhibitors of PARP may be able to attenuate ischemic and inflammatory cell and organ injury or may be able to enhance the cytotoxicity of antitumor agents. Both aspects of the "double-edged sword" role of PARP can be exploited for the experimental therapy of disease. As several classes of PARP inhibitors move towards clinical development, or have already entered the stage of clinical trials, we expect that in the upcoming few years, clinical proof of PARP inhibitors' therapeutic effect will be obtained in human disease. In the current short overview, we summarize the pros and cons and challenges with respect to the clinical use of PARP inhibitors, the expected clinical outcomes and potential risks. It appears that on the cytoprotective aspect of PARP, acute, life-threatening diseases (myocardial infarction, cardiopulmonary bypass in high-risk patients, and other, severe forms of ischemia-reperfusion to other organs including stroke and thoracoabdominal aneurysm repair) may represent some of the prime development indications. In the context of inhibition of DNA repair, combination of PARP inhibitors with certain antitumor agents (for example temozolomide) in patients with tumors with extremely poor prognosis are expected to provide the initial clinical results. Development of PARP inhibitors for additional indications (e.g. chronic use for the therapy of neurodegeneration and neuroinflammation, or diabetic complications) may be more challenging because of the unknown potential long-term side effects of PARP inhibitors.

Apoptosis↗
PubMed2001

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Binding Sites↗
PubMed2003

Single amino acid substitution enhances bacterial expression of PARP-4D214A.

Poly(ADP-ribose) polymerase-1 (PARP-1) is the canonical member of the PARP family of enzymes and modulates many crucial nuclear functions. PARP-1 is involved in apoptosis and is the substrate of caspase-3, a protease that cleaves PARP-1 at the conserved sequence 211DEVD214. To generate a caspase-3-uncleavable PARP-1, we introduced an amino acid substitution D214-->A214 at the site of cleavage. We observed that following over-expression in bacteria, the mutant protein HIS-PARP-1D214A was expressed several-fold more than a unmutated copy, HIS-PARP-1. The specific activity of HIS-PARP-1 enzyme in total bacterial extracts was 6.94 U/mg and 4.61 U/mg for HIS-PARP-1D214A. This approach should provide new avenues for crystallographic study of PARP-1 as well as new information for drug design targeting PARP-1.

Amino Acid Sequence↗
PubMed1997

Bcl-2 acts upstream of the PARP protease and prevents its activation.

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.

Journal Article↗
PubMed1999

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells.

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp(+/+)) ES cells and Parp(+/-) and Parp(-/-) ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp(-/-) clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp(+/-) clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp(-/-) clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Animals↗
PubMed2000

Differential susceptibility of normal and PARP knock-out mouse fibroblasts to proteasome inhibitors.

Recently we found a clearly reduced basal level of wt p53 protein in PARP-deficient cells. Interestingly, PARP deficiency affected only regularly spliced (RS) wt p53. No significant difference of the p53 transcription rate was observed between wt and PARP-lacking cells. To clarify whether the reduction of RS p53 protein is due to a lower translation rate or rather to its instability in the absence of functional PARP, we investigated the effect of the inhibition of proteasome activity and nuclear export on the p53 level. The p53 half-life was approximately eight-fold decreased in PARP-lacking cells. Surprisingly, treatment with three proteasome inhibitors increased RS p53 in normal but not in PARP-deficient cells. However, the inhibition of nuclear export resulted in a considerable accumulation of RS p53 in the latter. Therefore, we decided to increase concentrations of the inhibitors. Their higher concentrations strongly affected viability of normal, but not of PARP-deficient cells, about 70% of MEFs died. Interestingly, higher concentrations of proteasome inhibitors resulted in the appearance of RS p53 in PARP-lacking fibroblasts. Reconstitution of PARP-deficient cells with PARP restored the normal susceptibility to proteasome inhibitors thereby unequivocally demonstrating that the enhanced cytotoxicity of proteasome inhibitors and their action on p53 level depends on the presence of functional PARP.

Acetylcysteine↗
PubMed2003

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1.

We recently characterized the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1, whereas the amino-terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP-1 and overexpression of PARP-1. Our results unequivocally show that the presence of PARP-1 regulates the basal expression of wt p53 in unstressed cells.

Animals↗
PubMed2004

An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantitation of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors.

The enzyme poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP-1) catalyzes the formation of (ADP)-ribose polymers on a variety of protein acceptors in a NAD+ -dependent manner. While PARP-1 is activated by DNA damage and plays a critical role in cellular survival mechanisms, its overactivation leads to a depletion of NAD+/ATP energy stores and ultimately to necrotic cell death. Due to this dual role of PARP in the cell, small-molecule inhibitors of the PARP family of enzymes have been widely investigated for use as potentiators of anticancer therapies and as inhibitors of neurodegeneration and ischemic injuries. Unfortunately, standard assays for PARP inhibition are not optimal for the high-throughput screening of compound collections or combinatorial libraries. Described herein is a highly sensitive, inexpensive, and operationally simple assay for the rapid assessment of PARP activity that relies on the conversion of NAD+ into a highly fluorescent compound. We demonstrate that this assay can readily detect PARP inhibitors in a high-throughput screen using 384-well plates. In addition, the assay can be used to determine IC50 values for PARP inhibitors that have a range of inhibitory properties. As existing PARP assays utilize specialized reagents such as radiolabeled/biotinylated NAD+ or antibodies to poly(ADP-ribose), the chemical quantitation method described herein offers a highly sensitive and convenient alternative for rapidly screening compound collections for PARP inhibition.

Acetophenones↗
PubMed2005

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.

Androstadienes↗