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PubMed1992

The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters.

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

Base Sequence↗
PubMed2001

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells.

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.

Apoptosis↗
PubMed2005

Poly(ADP-ribose) polymerase (PARP-1) in homologous recombination and as a target for cancer therapy.

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumors. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumors.

Animals↗
PubMed1990

Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI).

A protein rich in proline and arginine (proline/arginine-rich protein (PARP] has been isolated from dissociative extracts of bovine nasal and articular cartilage, and its primary structure has been determined. The protein has 218 amino acids, giving a calculated protein Mr of 24,075. In nasal cartilage, this protein is in molar concentrations equivalent to 1/20-1/10 that of the link protein of cartilage proteoglycan aggregates. PARP has also been isolated from bovine articular cartilage, bovine fetal epiphysis, and nonossified human tarsal bones. PARP is similar to various collagen NH2-terminal domains. It is 49% identical to the NH2-terminal end of collagen alpha 1 (XI), 17% identical to the NC4 domain of collagen alpha 1 (IX), and 14% identical to the NC3 domain of collagen alpha 1 (XII). Four cysteines are conserved between type XI collagen and PARP, and these form two disulfide bonds. Two of the cysteines are also conserved between PARP and collagens IX and XII. The homology between the collagens and PARP makes it possible to speculate on the likely disulfide bond pattern in the collagen NH2-terminal domains. It is probable that PARP is a collagen fragment removed during processing in a manner analogous to chondrocalcin (the C-terminal propeptide of type II collagen).

Amino Acid Sequence↗
PubMed2000

Role of poly(ADP-ribose) polymerase (PARP) in DNA repair in sulfur mustard-exposed normal human epidermal keratinocytes (NHEK).

We previously reported that, in normal human epidermal keratinocytes (NHEK) cultures exposed to the alkylating compound sulfur mustard (bis-(2-chloroethyl) sulfide, HD, 0.3-1 mM), there is a rapid (< or =1 h) activation (100% above unexposed control) of the DNA repair enzyme DNA ligase I (130 kD) followed by a first-order decay (1-5 h). The DNA ligase activation is accompanied by a time-dependent (0.5-4 h) and significant DNA repair. Inhibition of another putative DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), by using 3-amino benzamide does not affect DNA ligase activation following HD exposure, but increases the half-life of the activated enzyme threefold. To examine the role of PARP in HD-induced DNA ligase activation and subsequent DNA repair, we conducted studies using cultured keratinocytes in which the level of PARP had been selectively lowered (> or =85%) by the use of induced expression of antisense RNA. In these cells, there was no stimulation of DNA ligase up to 3 h, and a small stimulation (ca. 30% above unexposed control at 5-6 h after HD exposure. A time-course (0.5-6 h) study of DNA repair in HD-exposed PARP-deficient keratinocytes revealed a much slower rate of repair compared with HD-exposed NHEK. The results suggest an active role of PARP in DNA ligase activation and DNA repair in mammalian cells, and also indicate that modulation of PARP-mediated mechanisms may provide a useful approach in preventing HD toxicity.

Cell Culture Techniques↗
PubMed1999

Compensatory expression of p73 in PARP-deficient mouse fibroblasts as response to a reduced level of regularly spliced wild-type p53 protein.

We have investigated the effect of PARP gene inactivation on the expression of wild-type (wt) p53 protein. Using immortalized fibroblasts from control and PARP knock-out mice we have found by immunoblotting with the PAb421 antibody a profound decrease of the p53 expression to a barely detectable level in PARP knock-out cells. Surprisingly, longer exposure of immunoblots revealed an immunoreactive band at about 75 kD which was stronger in PARP-deficient cells than in wt cells and was not affected upon doxorubicin treatment. The size of the PAb421 immunoreactive protein and the lack of its inducibility in response to DNA damage resembled those of p73, the first described p53 homologue. Therefore, we examined the reactivity of anti-p53 antibodies with in vitro translated p73 protein. Interestingly, p73 was efficiently immunoprecipitated with distinct antibodies recognizing the carboxy-terminus of p53. In Northern blots we observed p73 signals of comparable intensity in controls and PARP-deficient cells. We conclude that elevated expression of p73 may compensate the reduced level of p53 in PARP-deficient cells.

Animals↗
PubMed2001

Production, extraction, and purification of human poly(ADP-ribose) polymerase-1 (PARP-1) with high specific activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in a range of activities associated with DNA metabolism and plays a key role in maintaining the integrity of DNA and chromatin structure. As such, this enzyme is likely to provide a useful target when using a rational drug design approach to develop pharmaceutical reagents, including cancer therapeutics. However, there is still a great deal to learn about the mode of action of PARP-1 and therefore efforts are being directed at gaining a better understanding of the relationship between its structure and function. To this end we have developed a rapid and relatively simple approach to producing and purifying PARP-1. Unlike traditional PARP-1 purification protocols, the method described here requires only one chromatography step thus minimizing losses of the enzyme and also avoids the use of a competitive inhibitor-based affinity chromatography step, which is common to several other protocols in the literature. The product of the method described here is high-quality native PARP-1 with a high specific activity and K(m) and V(max) values similar to what is reported by other workers in the field. This protocol is particularly well suited to making PARP-1 in a quantity and of a quality suitable for structure-function studies.

Amino Acid Sequence↗
PubMed2004

Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans.

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Role of PARP on iNOS pathway during endotoxin-induced acute lung injury.

OBJECTIVE: Excessive nitric oxide (NO) and especially peroxynitrite may cause pulmonary tissue damage, e.g., through lipid peroxidation and/or exhaustion of cellular energy depletion induced by activation of poly (ADP-ribose) polymerase (PARP). Furthermore, PARP seems to aggravate tissue destruction by regulating the expression of respective genes. DESIGN: Prospective animal study. SETTING: University research laboratory. INTERVENTION: We investigated the effect of competitive PARP inhibition by 3-aminobenzamide (3-AB) on the pulmonary iNOS pathway after infusion of lipopolysaccharide (LPS). MEASUREMENTS AND RESULTS: The pretreatment of rabbits with 3-AB attenuated the LPS-induced iNOS mRNA and protein expression analyzed by RT-PCR and Western blot, and plasma nitrite concentrations quantified by Griess reaction (71+/-6%, 93+/-6% vs baseline). Electromobility shift assay showed an enhanced NF-kappaB and attenuated AP-1 activation after 3-AB vs LPS alone. Lipid peroxidation determined as levels of thiobarbituric acid reactive substances in plasma and lung tissue was reduced by 50% in the LPS+3-AB in comparison to LPS alone. Simultaneously, 3-AB was able to inhibit correspondingly the LPS-induced extravasation of gold-labeled albumin and increase of alveolo-arterial oxygen difference. CONCLUSION: PARP regulates the pulmonary NO pathway during endotoxemia via AP-1 and not NF-kappaB. Thus, pharmacological inhibition of PARP might be an effective intervention to prevent endotoxin-induced lung injury, interrupting the vicious circle of NO production and PARP activation.

Animals↗
PubMed2004

The PARP inhibitor benzamide protects against kainate and NMDA but not AMPA lesioning of the mouse striatum in vivo.

Overactivation of poly(ADP-ribose) polymerase (PARP) in response to genotoxic insults can cause cell death by energy deprivation. We previously reported that neurotoxic amounts of kainic acid (KA) injected into the rat striatum produce time-dependent changes in striatal PARP activity in vivo. Here, we have investigated the time-course of KA-induced toxicity and the effects of the PARP inhibitor benzamide on KA, AMPA and NMDA neurotoxicities in vivo, by measuring changes in the volume of the lesion and in NAD+ and ATP levels induced by the intra-striatal injection of these excitotoxins in C57Bl/6N mice. The KA-induced lesion volume was dependent on the amount of toxin injected and the survival time. The lesion was well developed at 48 h and was almost undetectable after one week. KA produced an extensive astrogliosis at one week. Benzamide partially prevented both KA- and NMDA- but not AMPA-induced lesions when measured at 48 h after the treatment. The effects of benzamide appeared to be in part related to changes in energy metabolism, since KA produced decreases in striatal levels of NAD+ and ATP that were partially prevented by benzamide at 48 h and which returned to control levels at one week. NMDA did not affect NAD+ and induced little alteration in ATP levels. Benzamide had no effect on AMPA-induced decreases in either NAD+ or ATP levels at 48 h. These results (1) indicate that PARP overactivation and energy depletion could be responsible in part for the cellular demise during the development of the lesion induced by KA; (2) confirm that PARP is involved in NMDA but not AMPA toxicities; (3) suggest the existence of differences between KA and AMPA-mediated toxicities; and (4) provide further evidence supporting PARP as a novel target for new drug treatments against neurodegenerative disorders.

Adenosine Triphosphate↗
PubMed2005

Parp-1 deficiency does not increase the frequency of tumors in the oral cavity and esophagus of ICR/129Sv mice by 4-nitroquinoline 1-oxide, a carcinogen producing bulky adducts.

The impact of poly(ADP-ribose) polymerase-1 (Parp-1)-deficiency on 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis was studied in mice with an ICR/129Sv mixed genetic background. Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) animals given 4NQO for thirty-two weeks at 0.001% in their drinking water developed papillomas and squamous cell carcinomas of the tongue, palate and esophagus, but with no statistically significant variation with the Parp-1 genotype. Thus Parp-1 deficiency does not elevate susceptibility to carcinogenesis induced by a carcinogen which gives rise to bulky DNA lesions. This study also indicated that the ICR/129Sv mixed genetic background is associated with high yield induction of esophageal tumors by 4NQO.

4-Nitroquinoline-1-oxide↗
PubMed2005

Synthesis and in vivo evaluation of [11C]PJ34, a potential radiotracer for imaging the role of PARP-1 in necrosis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme of eukaryotic cells that has been implicated in response to DNA injury. PARP-1 detects single-strand DNA breaks induced by a variety of genotoxic insults. A hyperactivation of PARP-1 is believed to play a critical role in tissues undergoing cellular death by necrosis. Therefore, a radiotracer that could image PARP-1 levels with PET could provide a useful tool in measuring necrosis in a variety of pathological conditions. The phenanthridinone derivative, 2-(dimethylamino)-N-(5,6-dihydro-6-oxophenanthridin-2-yl)acetamide (PJ34), has a high affinity for PARP-1 (IC(50) = 20 nM) and is a suitable lead compound for PET radiotracer development. The synthesis of [(11)C]PJ34 was accomplished by base-catalyzed reaction of the corresponding des-methyl precursor, N-(5,6-dihydro-6-oxophenanthridin-2-yl)-2-(methylamino)acetamide with [(11)C]methyl iodide in DMF. The radiolabeling yield was 60% and the specific activity was approximately 2000 mCi/micromol (decay corrected to E.O.B.). The total radiosynthesis time was approximately 50 min. Preliminary in vivo biodistribution studies in a rodent model of diabetes indicate that [(11)C]PJ34 displays a high uptake in tissues where PARP-1 is hyperactivated. These data indicate that [(11)C]PJ34 may be a useful radiotracer for imaging tissues undergoing cellular death via necrosis.

Animals↗
PubMed2005

Chemopotentiation by PARP inhibitors in cancer therapy.

Poly(ADP-ribose) polymerases (PARP) constitute a family of enzymes involved in the regulation of many cellular processes such as DNA repair, gene transcription, cell cycle progression, cell death, chromatin functions and genomic stability. Among the 18 members identified so far, PARP-1 and PARP-2 are the only proteins stimulated by DNA strand breaks and implicated in the repair of DNA injury. Therefore, these molecules have been exploited as potential targets for the development of pharmacological strategies to increase the antitumor efficacy of chemotherapeutic agents, which induce DNA damage. PARP inhibitors have been shown to restore sensitivity of resistant tumors to methylating agents or topoisomerase I inhibitors, drugs presently used for the treatment of primary and secondary brain tumors or malignancies refractory to standard chemotherapy. Interestingly, PARP inhibitors may also provide protection from the untoward effects exerted by certain anticancer drugs, which cause oxidative stress and consequent PARP overactivation. The aim of this article is to provide a brief overview of the recent literature on preclinical studies with the specific and potent inhibitors newly synthesized.

Animals↗
PubMed2003

Recombinant IFN-alpha2b treatment activates poly (ADPR) polymerase-1 (PARP-1) in KB cancer cells.

In the present paper, we investigated the relationship between the growth inhibitory effects of recombinant interferon-alpha2b (rIFN-alpha2b) and poly (ADPR) polymerase-1 (PARP-1) activity in the human squamous KB cancer cell line. Growth inhibition of the KB cells mediated by 1000 IU/ml of rIFN-alpha2b was accompanied by a transient rise in PARP-1 specific activity 24 h after rIFN-alpha2b treatment, confirmed by both the increase of intracellular poly (ADP-ribose) content and the PARP-1 auto-modification level. At longer times of incubation, the onset of apoptosis accompanied KB cell growth inhibition, as demonstrated by both flow cytometry and western-blotting analysis showing an 89 kDa apoptotic fragment of PARP-1. Moreover, pretreatment of the cells with the PARP-1 inhibitor, 3-aminobenzamide (3-ABA), at non-cytotoxic concentrations (1 mM), reduced the cell-growth inhibition, cell-cycle perturbation and apoptosis caused by rIFN-alpha2b. Taken together, these results strongly suggest that PARP-1 may be directly involved in the effects of rIFN-alpha2b in the KB cancer cell line.

Antineoplastic Agents↗
PubMed2003

Role of PARP under stress conditions: cell death or protection?

A great deal of increasing evidence designs PARP as a multifunctional protein implicated in many cellular functions. Much interest is emerging to understand the precise mechanisms by which PARP mediates genome stabilization and protection against damage, as well as its involvement in cell death, either apoptotic or necrotic. Aside from the clearly established role of PARP hyperactivation in necrotic cell death, after excessive DNA damage and energy failure, it appears to be actively involved in the phenomenon of apoptosis. However, its exact role is still controversial. The identification of several enzymes sharing the poly(ADP-ribose) polymerase catalytic domain (PARPs), but with different features and subcellular localization, has opened a new perspective in the field of poly(ADP-ribosyl)ation. The picture of the role of PARP in the control of cell homeostasis became even more complex after demonstration of its implication in the regulation of gene transcription. The notion that energy failure is the sole mechanism by which PARP promotes cell death is therefore under reevaluation.

Animals↗
PubMed1997

Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are DNA break-activated molecules, Although mice that lack PARP display no gross phenotype and normal DNA excision repair, they exhibit high levels of sister chromatid exchange, indicative of elevated recombination rates. Mutation of the gene for DNA-PK catalytic subunit (Prkdc) cases defective antigen receptor V(D)J recombination and arrests B- and T-lymphocyte development in severe combined immune-deficiency (SCID) mice. SCID V(D)J recombination can be partly rescued in T-lymphocytes by either DNA-damaging agents (gamma-irradiation and bieomycin) or a null mutation of the p53 gene, possibly because of transiently elevated DNA repair activity in response to DNA damage or to delayed apoptosis in the absence of p53. To determine whether the increased chromosomal recombination observed in PARP-deficient cells affects SCID V(D)J recombination, we generated mice lacking both PARP and DNA-PK. Here, we show that thymocytes of SCID mice express both CD4 and CD8 co-receptors, bypassing the SCID block. Double-mutant T-cells in the periphery express TCR beta, which is attributable to productive TCR beta joints. Double-mutant mice develop a high frequency of T-cell lymphoma. These results demonstrate that increased recombination activity after the loss of PARP anti-recombinogenic function can rescue V(D)J recombination in SCID mice and indicate that PARP and DNA-PK cooperate to minimize genomic damage caused by DNA strand breaks.

Animals↗
PubMed1999

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: evidence for increased tumor cell apoptosis in vivo.

Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.

Animals↗
PubMed2001

PARP co-activates B-MYB through enhanced phosphorylation at cyclin/cdk2 sites.

PARP is a multifunctional protein that can affect genome stability, transcription control, telomere length and cell death. Recently we have reported that PARP binds to and enhances B-MYB transactivating potential. B-MYB is a potentially oncogenic transcription factor involved in mammalian cell proliferation, survival and differentiation. B-MYB gene expression is growth regulated and B-MYB protein is phosphorylated during S phase by cyclin A or E/cdk2 kinase, resulting in augmented transactivating potential. Here we show that PARP induces phosphorylation of B-MYB protein at cdk2 phosphorylation sites, since a B-MYB protein with mutated cdk2 phosphorylation sites is refractory to PARP-induced phosphorylation and co-activation in mammalian cells. We propose that PARP functions as a B-MYB co-factor by promoting cyclin/cdk2-dependent B-MYB phosphorylation. These results highlight a novel role for PARP as a factor that integrates cyclin-dependent kinases signaling with gene transcription.

CDC2-CDC28 Kinases↗