Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.     

Involvement of poly (ADP-ribose) in the radiation response of mammalian cells

The evidence implicating poly (ADP-ribose) in the radiation response of mammalian cells is reviewed. It is concluded that the apparently conflicting results using inhibitors of ADP-ribosyl transferase (ADPRT) can be explained by a working hypothesis. This hypothesis maintains that poly (ADP-ribose) is required for repair of radiation damage (presumably to facilitate ligation). In most cells the synthesis of poly (ADP-ribose) is not rate limiting for repair and therefore, an almost complete inhibition of ADPRT activity is required to potentiate the radiation response. In radiation-sensitive cells (e.g. resting lymphocytes, L5178Y-S cells) with a deficient poly (ADP-ribose) metabolism, its synthesis can become rate limiting for repair. In such cells even a partial inhibition of ADPRT activity may enhance radiation-induced cell killing. It is suggested that if such differences exist between normal and cancer cells, they can be utilized to improve the therapeutic ratio of radiotherapy.     

Response of V79 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment: inhibition of poly(ADP-ribose) and topoisomerase activity.

MNNG-induced killing of V79 cells has been found to be enhanced on inhibition of topoisomerase II activity by nalidixic acid and poly(ADP-ribose) polymerase synthesis by benzamide. Using these 2 inhibitors in conjunction after MNNG treatment, some overlap in the functions of these 2 enzymes was observed. Nalidixic acid and benzamide were found to suppress the yields of mutations and SCEs induced by MNNG. Benzamide was more effective in suppressing the mutation yield whereas nalidixic acid was more effective in suppressing SCEs. A model based on the relative requirement of topoisomerase and poly(ADP-ribose) for the repair of different types of damage has been proposed to explain the results.     

Potentially lethal damage, deficient repair in X-ray-sensitive caffeine-responsive Chinese hamster cells

Further studies are described with a radiation-sensitive clone of V79 Chinese hamster cells, designated V79-AL162/S-10. Extended postirradiation treatment with caffeine causes V79-AL162/S-10 cells to respond like repair-competent V79 cells, but both kinds of cells suffer enhanced killing by caffeine, in a similar fashion, when the postirradiation treatment period is relatively brief. Extended postirradiation treatment of repair-competent cells causes them to respond like sensitive cells without caffeine post-treatment. Treating irradiated V79-AL162/S-10 cells with hypertonic saline appreciably reduces the survival rescue which can be effected by caffeine. This latter observation leads to the inference that the sectors of damage affected by anisotonic shock and caffeine post-treatment overlap. From these and other results we propose that the DNA replicational machinery of the cell is the locus of action of these radiation damage/repair processes.     

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.     

Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.     

Poly(ADP-ribose) and the recovery from damage in Chinese hamster cells due to 5-bromodeoxyuridine photolysis

Exposure of Chinese hamster cells to near-u.v. light, following the uniform incorporation of 5-bromodeoxyuridine (BrdUrd) into their DNA, resulted in cell killing that was close to exponential. An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3-ABA), enhanced the cytotoxic effect of this treatment when present for 2 h at 20 mM after light exposure. The dose modifying factor was 1.4. Under conditions that resulted in a sigmoidal survival curve (a 30 min BrdUrd pulse in S phase, followed 90 min later by light exposure) the effect of 3-ABA was to remove the shoulder of the survival curve with very little change in its final slope. Using various inhibitors of ADP-ribosyl transferase (ADPRT) the enhanced cell killing was found to correlate with the inhibitors' relative potency. Cellular NAD+, the substrate for poly(ADP-ribose) synthesis, was rapidly depleted after exposure. This depletion was largely prevented by 3-ABA; the activity of ADPRT increased with the fluence of near-u.v. light; and the concentration of cellular NAD+ decreased with exposure. ADPRT activity was maximal immediately after exposure to near u.v. light and then decayed to pre-exposure levels within 30 min (37 degrees C). The enhanced cytotoxicity of BrdUrd + near-u.v. light, when followed by 3-ABA treatment, disappeared at a rate similar to that of the decay in ADPRT activity. We conclude from these results that poly(ADP-ribose) synthesis is important for the recovery from BrdUrd photolysis damage in DNA. Because this damage and its repair are relatively specific (e.g. compared to ionizing radiation) and relatively easy to manipulate, it could serve as a model system for the study of the role of poly(ADP-ribose) in the repair of DNA damage.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Influence of poly(ADP-ribose) synthesis inhibitors on the repair of sublethal and potentially lethal damage in gamma-irradiated mammalian cells

The influence of poly(ADP-ribose) synthesis inhibitors on mammalian cell radiosensitivity was investigated. Four different inhibitors were studied: 3-methoxybenzamide, 3-aminobenzamide, 6-aminonicotinamide and nicotinamide. When exponentially growing or plateau phase cells are incubated before irradiation with non-toxic concentrations of these compounds, their radiosensitivity is enhanced except in the case of nicotinamide. The poly(ADP-ribose) inhibitors do not modify the repair of sublethal damage, but totally suppress the repair of potentially lethal damage, as shown by the survival of CHO cells and of a human osteosarcoma cell line.     

Potentiation of cell killing by inhibitors of poly(ADP-ribose) polymerase in four rodent cell lines exposed to N-methyl-N-nitrosourea or UV light

The sensitivities (Do-values) of the cytotoxic effect of MNU on four rodent cell lines were: mouse L1210, 0.07 mM; rat Yoshida sarcoma, 0.52 mM; Chinese hamster V79A, 0.70 mM and the UV sensitive, X-ray sensitive V79/79, 0.35 mM. The abilities of maximum non-toxic doses of the poly-(ADP-ribose) polymerase inhibitors, 5-methyl nicotinamide (5MeN), 3-methoxybenzamide (3MBA) and caffeine to potentiate this cytotoxicity and that of UV light in V79A and V79/79 was measured. The degree of potentiation (ratio Do without inhibitor/Do with inhibitor) was both agent and cell line dependent. In general the lymphoid cell lines L1210 and YS showed greater potentiation, up to 4-fold, than did the fibroblast lines V79A and V79/79. The use of inhibitors in pairs suggested that 5MeN and 3MBA affect one process whereas caffeine affects additional processes. The data provide further support for a role for poly(ADP-ribose) in DNA repair, but indicate that metabolic factors may modify the effectiveness of individual inhibitors of poly(ADP-ribose) polymerase in different cell lines.     

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.     

Effect of PD 128763, a new potent inhibitor of poly(ADP-ribose) polymerase, on X-ray-induced cellular recovery processes in Chinese hamster V79 cells

The modifying effects of PD 128763 (3,4-dihydro-5-methyl-1(2H)-isoquinolinone), a potent inhibitor of poly(adenosine-diphosphate (ADP)-ribose) polymerase, on radiation-induced cell killing were examined in Chinese hamster V79 cells. This compound has an IC50 value against the purified enzyme approximately 50X lower than 3-aminobenzamide (3-AB), a widely used specific inhibitor of the enzyme. Exposure of exponentially growing cells to a noncytotoxic concentration (0.5 mM) of PD 128763 for 2 h immediately following X irradiation increased their radiation sensitivity, modifying both the shoulder and the slope of the survival curve. When recovery from sublethal damage and potentially lethal damage was examined in exponential and plateau-phase cells, respectively, postirradiation incubation with 0.5 mM PD 128763 was found not only to inhibit both these processes fully, but also to enhance further the level of radiation-induced cell killing. This is in contrast to the slight effect seen with the less potent inhibitor, 3-AB. The results presented suggest that the mechanism of radiosensitization by PD 128763 is related to the potent inhibition of poly(ADP-ribose) polymerase by this compound.     

Cellular responses to ionizing radiation: effects of interrupting DNA repair with chemical agents

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is 'fixed' by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells

The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks.     

Bleomycin induces similar survival variations through the cell cycle as do X rays with and without hypertonic shock.

In an earlier report [H. Utsumi and M. M. Elkind, Radiat. Res. 119, 534-541 (1989)], it was shown that the survival of V79 Chinese hamster cells treated with bleomycin was significantly reduced by a posttreatment with anisotonic phosphate-buffered saline in a manner that was qualitatively similar to what had been observed with X rays [H. Utsumi and M. M. Elkind, Radiat. Res. 77, 346-360 (1979)]. This similarity suggested that similarities might exist in the cyclic variation in the suppression of the repair of potentially lethal damage following treatment with bleomycin or X rays. Accordingly, the age-response variations of survival, with or without a posttreatment challenge with hypertonic buffer, were compared in the same experiment when cells were treated with either agent. Although a significant difference was observed near the G1/S-phase border, in general the damage induced by the two agents showed a similar dependence on cell age, and posttreatment with hypertonic buffer enhanced cell killing appreciably following either treatment. The results support the inference that bleomycin is a radiomimetic agent.     

Targeting poly(ADP-ribosyl)ation: a promising approach in cancer therapy

Recent progress in the field of DNA repair has demonstrated that transient inhibition of DNA damage detection or repair using potent poly(ADP-ribose) polymerase (PARP) inhibitors could improve the efficacy of cancer treatments. Although more study is needed, recent publications lead to optimism that the inhibition of poly(ADP-ribose) synthesis could selectively kill cancer cells when used to treat tumours with defective BRCA proteins. These reports and others shed some light on the DNA damage signalling and repair processes involving PARPs. However, a better understanding of the molecular mechanisms regulated by poly(ADP-ribose) metabolism will be essential before optimism can be replaced by clinical realization.     

The effect of nicotinamide on N-methyl-N''-nitro-N-nitrosoguanidine induced killing and mutation

Nicotinamide, an inhibitor of poly(ADP-ribose) synthesis has been found to increase the killing of Chinese hamster V-79 cells induced by N-methyl-N′-nitro-N-nitrosoguanidine in concentration dependent manner. Such treatment also decreased the induced mutation. The nature of the results obtained was similar to those obtained earlier with benzamide, another inhibitor.     

Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.     

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.     

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]