Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.     

Isolation of cDNA encoding full-length rat (Rattus norvegicus) poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP-ribose). Poly(ADP-ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation. We established the complete cDNA sequence of rat poly(ADP-ribose) polymerase by RT-PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules of poly(ADP-ribose) polymerase.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.     

Modulation of (+/-)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.05-1 microM (+/-)-anti-BPDE resulted in elevated p53 protein levels as compared to the constitutive levels of control cells. The rapid induction response, detectable within a few hours, was sustained up to a period of at least 24 h. Repair-proficient and repair-deficient (XPA) human lymphoblastoid cells showed a similar response. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction response by concomitantly decreasing the extent of (+/-)-anti-BPDE induced DNA damage in cells pretreated with the inhibitor. However, the direct involvement of poly ADP-ribosylation was also apparent as 3-AB was able to attenuate (approximately 50%) the p53 response by post-damage inhibitor treatment of the cells. Inhibition of cellular DNA replication by hydroxyurea and AraC, in the presence or absence of DNA damage, also resulted in rapid p53 accumulation in repair-deficient cells. On the contrary, inhibition of protein kinase C (PKC) by calphostin-C led to an abrogation of (+/-)-anti-BPDE mediated p53 induction. Analysis of the downstream effects of carcinogen treatment showed that the lymphoblastoid cells undergo DNA fragmentation indicative of apoptosis while fibroblasts exhibit cell cycle arrest at the G1-S boundary.     

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1

BACKGROUND: Exogenous surfactant therapy of lung donors improves the preservation of normal canine grafts. The current study was designed to determine whether exogenous surfactant can mitigate the damage in lung grafts induced by mechanical ventilation before procurement. METHODS AND RESULTS: Five donor dogs were subjected to 8 hours of mechanical ventilation (tidal volume 45 ml/kg). This produced a significant decrease in oxygen tension (p = 0.007) and significant increases in bronchoscopic lavage fluid neutrophil count (p = 0.05), protein concentration (p = 0.002), and the ratio of poorly functioning small surfactant aggregates to superiorly functioning large aggregates (p = 0.02). Five other animals given instilled bovine lipid extract surfactant and undergoing mechanical ventilation in the same manner demonstrated no significant change in oxygen tension values, lavage fluid protein concentration, or the ratio of small to large aggregates. All 10 lung grafts were then stored for 17 hours at 4 degrees C. Left lungs were transplanted and reperfused for 6 hours. After 6 hours of reperfusion the ratio of oxygen tension to inspired oxygen fraction was 307 +/- 63 mm Hg in lung grafts administered surfactant versus 73 +/- 14 mm Hg in untreated grafts (p = 0.007). Furthermore, peak inspired pressure was significantly (p < 0.05) lower in treated animals from 90 to 360 minutes of reperfusion. Analysis of lavage fluid of transplanted grafts after reperfusion revealed small to large aggregate ratios of 0.17 +/- 0.04 and 0.77 +/- 0.17 in treated versus untreated grafts, respectively (p = 0.009). CONCLUSIONS: Instillation of surfactant before mechanical ventilation reduced protein leak, maintained a low surfactant small to large aggregate ratio, and prevented a decrease of oxygen tension in donor animals. After transplantation, surfactant-treated grafts had superior oxygen tension values and a higher proportion of superiorly functioning surfactant aggregate forms in the air space than untreated grafts. Exogenous surfactant therapy can protect lung grafts from ventilation-induced injury and may offer a promising means to expand the donor pool.     

Effect of a poly(ADP-ribose) polymerase inhibitor on DNA breakage and cytotoxicity induced by hydrogen peroxide and gamma-radiation

Thirty-six outpatients aged 20 to 51 with RDC primary major depressive disorder (MDD) completed a 5-week trial of desipramine following a week of single-blind placebo. Five had a past history of hypomanic disorder. For all but one patient, daily dosage at bedtime was constant for the final 4 weeks, with a mean (S.D.) of 168.1 (46.5) mg. Plasma samples drawn at the three final weekly visits were assayed by high-performance liquid chromatography for 2-hydroxydesipramine (2-OH-DMI) and desipramine. Mean (S.D.) plasma levels were 59.8 (30.0) ng/ml for 2-OH-DMI and 142.9 (138.6) ng/ml for desipramine. Thirteen patients (36%) had a final 17-item Hamilton depression rating < and = 6 and were classified as responders. According to receiver operating characteristics analysis, patients with plasma 2-OH-DMI levels > and = 58 and < 92 ng/ml had a greater likelihood of responding than those with lower or higher levels (p = 0.005, Fisher's exact test), while patients with plasma desipramine levels > and = 64 ng/ml were more likely to respond than those with lower levels (p = 0.032, Fisher's exact test). Results using an alternate response criterion were similar. These findings suggest that in desipramine-treated outpatients with primary MDD the relationship between therapeutic response and plasma levels is curvilinear for 2-OH-DMI and linear for desipramine.     

Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.     

Poly (ADP-ribose) polymerase, a potential target for drugs: Part II. Regulation of differentiation by the poly ADP-ribose system

It is shown that eukaryotic differentiation is specifically sensitive to pADPRT regulation in Trypanosomna, Leishmania and Mytilus models. There is powerful inhibition of early differentiation without cell toxicity by pADPRT ligands.     

Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.     

Induction of hepatic poly(ADP-ribose) polymerase by peroxisome proliferators, non-genotoxic hepatocarcinogens

The binding capacity of FK506 binding protein (FKBP) was examined after 2-h hemispheric ischemia in the gerbil brain in order to clarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal gerbil brain using 1 nM [3H]dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner, although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites of FK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an ischemia group (n = 12) and a sham group (n = 6). The right common carotid artery was ligated to induce hemispheric ischemia for 2 h in the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the [14C]iodoantipyrine method. The ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 ml/100 g/min were utilized as the ischemia group (n=6) for further data analysis. No significant differences in FK506 binding between the ischemia and sham groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-h ischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase of cerebral ischemia.     

Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death

Proteases play a crucial role in apoptosis or programmed cell death. The aim of this review is to highlight the purpose for which these proteases are activated, i.e., to specifically cleave a select subset of cellular proteins at an appropriate time during cell death. Poly(ADP-ribose) polymerase (PARP), a nuclear protein implicated in DNA repair, is one of the earliest proteins targeted for a specific cleavage to the signature 89-kDa fragment during apoptosis. Characterization of the apoptotic cleavage of PARP and other target proteins helped in understanding the role of cysteine aspartic acid specific proteases (caspases) in the apoptotic process. We have recently identified that in some models of cell death, the cleavage pattern for PARP is different from production of the signature 89-kDa fragment. Necrotic death of HL-60 cells and apoptotic death of Jurkat cells mediated by granzyme B and perforin were accompanied by distinct additional fragments, suggesting cleavage of PARP at other sites by caspases or other death proteases. This review summarizes how detection and characterization of PARP cleavage could serve as a sensitive parameter for identification of different types of cell death and as a marker for activation of different death proteases. The putative biological functions for early cleavage of PARP in apoptosis are also discussed.     

Identification of commonly recognized epitopes on a rare autoantigen: poly(ADP-ribose) polymerase

To analyze mechanisms of autoantibody production, epitope mapping of a rare autoantigen, poly(ADP-ribose) polymerase, was performed. A cDNA fragment (1873 bp long), which was already confirmed to encode the autoepitopes of this protein, was subcloned into a protein expression plasmid pEX. Several deletion mutants were produced by enzymatic treatments of this construct. PCR-amplified cDNA fragments were also individually subcloned into this vector. The recombinant proteins produced in Escherichia coli by these vectors were tested for their respective antigenicities by immunoblotting. It was found that all positive sera tested (seven cases) strongly recognized common epitopes in a restricted region of the molecule. Furthermore, three out of the seven positive sera were found to recognize other parts of the molecule. The data suggest possible mechanisms for the formation of anti-poly(ADP-ribose) polymerase autoantibodies.     

Antitumor agents. Part 186: Synthesis and biological evaluation of demethylcolchiceinamide analogues as cytotoxic DNA topoisomerase II inhibitors

Demethylation of colchiceinamide (2) and its analogues (3-10) afforded a novel class of mammalian DNA topoisomerase II inhibitors (2a-10a) without displaying tubulin inhibitory activity. All target compounds inhibited the catalytic activity of topoisomerase II at drug concentrations at 100 microM. An in vitro cytotoxicity assay indicated that compounds 3a and 8a were strong and tissue-selective cytotoxic agents against the MCF-7 breast cancer cell line (IC50 = 0.36 and 0.48 microgram/mL, respectively) and the CAKI-1 renal cancer cell line (IC50 = 0.72 and 0.96 microgram/mL, respectively).     

Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching

Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.     

Inhibitor and NAD+ binding to poly(ADP-ribose) polymerase as derived from crystal structures and homology modeling

Inhibitors of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) are of clinical interest because they have potential for improving radiation therapy and chemotherapy of cancer. The refined binding structures of four such inhibitors are reported together with the refined structure of the unligated catalytic fragment of the enzyme. Following their design, all inhibitors bind at the position of the nicotinamide moiety of the substrate NAD+. The observed binding mode suggests inhibitor improvements that avoid other NAD(+)-binding enzymes. Because the binding pocket of NAD+ has been strongly conserved during evolution, the homology with ADP-ribosylating bacterial toxins could be used to extend the bound nicotinamide, which is marked by the inhibitors, to the full NAD+ molecule.     

Synthesis and biological properties of 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil

A novel 5-o-carboranyl-containing nucleoside, 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (6, CFAU), was synthesized as a potential intracellular neutron capture agent. This compound was prepared in five steps starting from 5-iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (1). The desired carboranyl derivative was obtained by addition of decaborane [as the bis(propionitrile) adduct] to the protected acetylenic nucleoside precursor followed by debenzoylation. The synthesis of CFAU was also performed by glycosylation of the suitably protected 5-o-carboranyluracil with the appropriate 2-fluoroarabinosyl derivative. This compound was evaluated for its cytotoxicity in human lymphocytes, monkey cells, and rat and human gliomas cells, as well as for antiviral activity against human immunodeficiency virus and herpes simplex virus type 1. Its biological activity was compared to 5-o-carboranyl-1-(2-deoxyribofuranosyl)uracil in these cell culture systems, human bone marrow cells, and mice. The results obtained to date suggest that CFAU has suitable characteristics as a sensitizer for boron neutron capture therapy.     

Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion

Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.