Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP-ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP-ribose) DNA strand break DNA repair     

Constraints to DNA unwinding near radiation-induced strand breaks in Ewing's sarcoma cells

Ewing's sarcoma cell lines were compared to other cell lines for induction of DNA strand breaks by ionizing radiation and their ability to repair those breaks. The alkali-unwinding assay and alkaline sucrose gradient analysis were used for these studies. The alkali-unwinding assay revealed that the amount of DNA unwound per strand break in Ewing's sarcoma cells was less than for other cells and was not influenced by high-salt denaturation conditions. Ewing's sarcoma cells had similar induction and repair rates for strand breaks compared with other cell lines. The kinetics of unwinding suggests there are constraints to DNA unwinding in the chromatin of Ewing's sarcoma cells, possibly related to high levels of poly(ADP-ribose) polymerase in these cells.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.     

Different effects of tert-butylhydroperoxide-induced peroxynitrite-dependent and -independent DNA single-strand breakage on PC12 cell poly(ADP-ribose) polymerase activity.

The short-chain lipid hydroperoxide analogue tert-butylhydroperoxide induces peroxynitrite-dependent and -independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert-butylhydroperoxide treatment with peroxynitrite-independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP-ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP-ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP-ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert-butylhydroperoxide-induced peroxynitrite-independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP-ribose) polymerase.     

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

Poly(ADP-ribose) metabolism in ultraviolet irradiated human fibroblasts

Exposure of human fibroblasts to 5 J/m2 of UV light resulted in a rapid increase of up to 1500% in the intracellular content of poly(ADP-ribose) and a rapid depletion of its metabolic precursor, NAD. When added just prior to UV treatment, the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, totally blocked both the increase of poly(ADP-ribose) and decrease in NAD for up to 2.5 h. Addition of 3-aminobenzamide at the time of maximal accumulation of poly(ADP-ribose) resulted in a decrease to basal levels with a half-life of approximately 6 min. The rates of accumulation of poly(ADP-ribose) and depletion of NAD were increased in the presence of either 1-beta-arabinofuranosylcytosine or hydroxyurea. Since these agents are known to cause an additional accumulation of DNA strand breaks following UV irradiation, these data provide evidence for a mechanism in which the rate of poly(ADP-ribose) synthesis following DNA damage is regulated in intact cells by the number of DNA strand breaks. Under conditions in which the synthesis of poly(ADP-ribose) was blocked, DNA repair replication induced by UV light was neither stimulated nor inhibited.     

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.     

Baseline sister chromatid exchange in human cell lines with different levels of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.     

Temporal Patterns of Poly(ADP-Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.     

Poly(ADP-ribose) and the response of cells to ionizing radiation

The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.     

Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

DNA strand break metabolism in human lymphocytes: a reevaluation

Recent findings concerning the presumed existence of single-strand breaks (SSB) in quiescent human peripheral blood lymphocytes (PBL) are discussed in relation to the role of poly(ADP-ribosyl)ation in DNA strand break metabolism. It is argued that the activation of poly(ADP-ribose)polymerase (ADPRP) by a DNA-damaging agent is not indicative of an obligatory role of poly(ADP-ribosyl)ation in DNA repair. From this it follows that SSB induced by different strand-breaking agents might be removed by either ADPRP-dependent or ADPRP-independent DNA repair pathways.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

DNA strand breaks, NAD metabolism, and programmed cell death

An intimate relationship exists between DNA single-strand breaks, NAD metabolism, and cell viability in quiescent human lymphocytes. Under steady-state conditions, resting lymphocytes continually break and rejoin DNA. The balanced DNA excision-repair process is accompanied by a proportional consumption of NAD for poly(ADP-ribose) synthesis. However, lymphocytes have a limited capacity to resynthesize NAD from nicotinamide. An increase in DNA strand break formation in lymphocytes, or a block in DNA repair, accelerates poly(ADP-ribose) formation and may induce lethal NAD and ATP depletion. In this way, the level of DNA single-strand breaks in the lymphocyte nucleus is linked to the metabolic activity of the cytoplasm. The programmed removal of lymphocytes (and perhaps of other cells) with damaged DNA, may represent a novel physiologic function for poly(ADP-ribose)-dependent NAD cycling.     

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.